In the U.S. state of Delaware, U.S. Route 301 runs from U.S. Route 40 in Glasgow to the Maryland state line where it continues as the Blue Star Memorial Highway. The route is mostly a two-lane undivided highway, however there are plans to move it onto a new tolled highway to better connect northern Delaware with Annapolis and points south along Interstate 95, without the need to pass through Baltimore.

US 301 enters Delaware from Maryland to the east of Warwick, Maryland, where it narrows from the four-lane, divided Blue Star Memorial Highway in Maryland to the two-lane undivided Middletown Warwick Road in Delaware. The road heads through a mix of farms and woods, coming to an intersection with DE 299. At this point, DE 299 forms a concurrency with US 301, with the road passing through more agricultural areas with some development and widening into a four-lane divided highway. The road comes to an intersection with DE 15, where that route joins US 301/DE 299. The road enters Middletown and heads into commercial areas. At an intersection, the three routes split, with DE 15 heading northwest on Bunker Hill Road, DE 299 heading east on Main Street, and US 301 continuing north along Middletown Warwick Road.

US 301 passes more businesses before heading north on Summit Bridge Road. The road narrows into a two-lane undivided road and passes through a mix of fields and residential and commercial development as it runs immediately to the west of Norfolk Southern's Delmarva Secondary railroad line, reaching an intersection with DE 71. Here, DE 71 heads north concurrent with US 301, with the road passing near some businesses and a housing development before leaving Middletown. The road runs a short distance to the west of the Norfolk Southern rail line as it continues farmland with some scattered residential and commercial development. In Mount Pleasant, DE 896 joins US 301/DE 71 from the east and the road widens from a two-lane undivided road to a four-lane divided highway. The road passes a few businesses before heading between the Summit Airport to the west and housing developments to the east. US 301/DE 71/DE 896 heads to the northwest through farm fields before turning to the west and passing a residential subdivision to the south. In the unincorporated community of Summit Bridge, the road intersects the northern terminus of DE 15. Past this intersection, US 301/DE 71/DE 896 turns north and crosses over the Chesapeake and Delaware Canal on the Summit Bridge.

Past the Summit Bridge, the road passes through woods and passes under Chesapeake City Road near the Delaware Veterans Memorial Cemetery. DE 71 splits from US 301/DE 896 by heading to the southeast on Red Lion Road. US 301/DE 896 continues north between residential neighborhoods to the west and wooded Lums Pond State Park to the east. The road passes near more suburban homes with some fields before reaching Glasgow, where it curves northeast to bypass Glasgow to the east with DE 896 Bus. heading north into Glasgow. US 301/DE 896 curves northwest and intersects US 40, where US 301 comes to its northern terminus and DE 896 continues to the north.

US 301 in Delaware has an annual average daily traffic count ranging from a high of 38,941 vehicles at the northern terminus at US 40 to a low of 14,254 vehicles at the Peterson Road intersection in Middletown. The entire length of US 301 in Delaware is part of the National Highway System.

U.S. Route 301 was first signed in Delaware in 1960, when it was rerouted from its northern terminus in Baltimore, Maryland to follow U.S. Route 50 from Bowie, Maryland and over the Chesapeake Bay Bridge to Queenstown, Maryland. US 301 then replaced the four-lane, divided Maryland Route 71 between Queenstown and the Delaware border. It then followed Delaware Route 71 to Middletown, where northbound and southbound US 301 split. Northbound US 301 followed Delaware Route 299 east to Odessa and then U.S. Route 13 north to Interstate 295 at Farnhurst, crossing the Chesapeake and Delaware Canal on the St. Georges Bridge. Southbound US 301 followed US 13 from Farnhurst south to Red Lion. It then followed Delaware Route 71 south to Middletown, crossing the Chesapeake and Delaware Canal on the Summit Bridge. In the 1970s, the alignment of northbound US 301 was changed to follow DE 71 over the Summit Bridge and then Delaware Route 896 north to Glasgow. It then headed east on U.S. Route 40 to State Road where it resumed north on US 13 to Farnhurst. In the 1980s, US 301 was moved to one alignment which followed Delaware Route 896 between DE 71 in Mount Pleasant and US 13 in Boyds Corner and US 13 from Boyds Corner to Farnhurst. Due to structural problems along the St. Georges Bridge, a truck route, U.S. Route 301 Truck was created, following Delaware Route 896 between Mount Pleasant and Glasgow and US 40 between Glasgow and State Road. In 1992, AASHTO approved relocating US 301 to its present alignment which ends at US 40 in Glasgow.

The U.S. Route 301 Project is a proposed upgrade, which will replace US 301 with a 17.4-mile (28.0 km), four-lane toll road connecting the existing four-lane U.S. Route 301 in Maryland near Warwick, Maryland to the Delaware Route 1 toll road near St. Georges, Delaware. The cost for the project is estimated to be $738 million. The road will serve as a high-speed bypass around the cities of Middletown and Newark, Delaware and has a proposed construction start date in mid-2012.

On November 13, 2006, DelDOT announced that it has chosen the "green route + spur" option to build the new US 301. The main highway itself will consist of a four-lane, limited access toll road built to the same Interstate Highway standards as that of Route 1 (either a concrete surface with asphalt shoulders or, as a time-saving measure, an all-asphalt surface like that used on the Puncheon Run Connector in Dover) and will have exits for two local roads, as well as Delaware Route 299, the current two-lane U.S. 301 (which is also signed as Delaware Route 71), and Delaware Route 896. A two-lane limited access spur, will connect the main highway with the Summit Bridge at the intersection with Delaware Rts. 71/896 and Delaware Route 15, which connects the Summit Bridge with Chesapeake City, Maryland (via Delaware Route 286).

The northern terminus of the highway will be at Route 1 between the Chesapeake and Delaware Canal Bridge and the Biddle's Corner toll plaza, most likely using the existing one-way exit that was constructed in 2000 to provide access to U.S. 13 in South St. Georges, but with provisions to provide complete access to U.S. 301 from Del. Rt. 1 without having to use Del. Rt. 896 as a connector. The "green + spur" option is the lowest cost, and will have minimum impact on property acquisition (35 residential and business properties total, mostly farmland) and would not allow for the demolishing of two local churches. Thus the location of the exit between the bridge and the toll plaza will allow the U.S. 301 upgrade to serve as a sort-of "Chesapeake Bay Extension" of the Route 1 as well as bringing the original bi-state toll road project back to full circle – the building of two separate toll roads connecting the Washington D.C. metro area with the New Jersey Turnpike, except that the Maryland portions are currently not tolled except at its Susquehanna River/Chesapeake Bay crossings.

This proposal is opposed by many in the area, most notably the Middletown Corridor Coalition. The MCC is opposed to the "Spur" on the Rt 301 project feeling that it was added in mid-workshop process and the public was given no alternatives to the Spur. The MCC is working with area legislators to "uncouple" the Spur from the Project and have it as a "stand alone" project. It remains to be seen if this 135 million dollar addition will make it through the ROD process by the FHWA. There is also the matter of whether traffic trends and projected toll-revenue will warrant the need for the project. Projections of traffic volume and revenue reported in 2011 are substantially below those reported in mid-2010 and projections on which decision making in 2008 were made.

Plans for a four-lane superhighway connecting the New Jersey Turnpike (via the Delaware Memorial Bridge) with the Washington, D.C. metro area dates back to the opening of the Chesapeake Bay Bridge in the mid-1950s. As to provide adequate coverage for the entire Chesapeake Bay region, two separate toll highways, to be jointly operated by both Maryland and Delaware, were proposed. One road would parallel the existing U.S. 301 (then a two-lane road), but this was never built, although U.S. 301 was eventually upgraded to a high-speed four-lane road with both at-grade and grade-separated exits. Another road, to be part of the mainline I-95 route between Maine and Florida, was eventually built, as the John F. Kennedy Memorial Highway and the Delaware Turnpike, and followed the western shore of the Chesapeake Bay to Baltimore and Washington, D.C., with a spur (I-97) providing access to Annapolis, Maryland. Despite the upgrade of most of U.S. 50 and 301 to freeway standards between Washington, D.C. and Queen Anne's County (which included the building of a parallel span to the Chesapeake Bay Bridge) and upgrading U.S. 301 to four lanes, limited budgets in Delaware, along with local opposition from farmers, prevented the state upgrading U.S. 301 to a four-lane road, although in 1960, the U.S. Army Corps of Engineers, during a major widening and upgrade of the Chesapeake and Delaware Canal to its present configuration, built the new four-lane Summit Bridge to freeway standards, with its approaches using a 3% climbing grade instead of the 5% grade used on the nearby Chesapeake City and St. Georges Bridges.

Despite the building of the Summit Bridge, U.S. 301 remained, for most of the last 40 years, as either a two-lane road, or as part of U.S. 13 or U.S. 40 (U.S. 301, which currently ends in Glasgow, originally began at the Delaware Turnpike's Farnhurst interchange in New Castle). In the 1980s, in an effort to provide a bypass around Newark, Delaware, DelDOT proposed building a four-lane highway, signed as U.S. 301, that would bypass Glasgow, connect with the Delaware Turnpike (I-95) south of Newark, and then branch off between the current Delaware Route 896 interchange and the service plaza, via an unbuilt interchange (this is why there is no Exit 2 on I-95). Because of local opposition, DelDOT, in the 1990s, upgraded the mostly two-lane U.S. 301/Route 896 highway between Boyds Corner and Glasgow to a four-lane divided highway with surface intersections, with a bypass around Glasgow to connect U.S. 301 with U.S. 40, itself also being a surface intersection.

With the surrounding area around Middletown undergoing exponential growth in the 1990s and early 2000s, along with traffic tieups on U.S. 301 and neighboring state highways being common, DelDOT investigated in the resurrecting of the U.S. 301 highway project, but instead of using the original plans in the 1960s, DelDOT instead looked into connecting U.S. 301 with the new Route 1 in St. Georges. In addition, because of the cuts in federal funds to build and maintain highways, along with U.S. 301 being used as a major heavy truck route, DelDOT also looked into building a toll plaza on the Delaware/Maryland State Line (similar to the Newark Toll Plaza on the Delaware Turnpike) in order to collect the needed tolls to fund the costs of the new road.

The entire route is in New Castle County.