It goes through the cities of Middletown, Delaware; Annapolis, Maryland; Richmond and Petersburg, Virginia; Rocky Mount, Wilson, and Fayetteville, North Carolina; Florence, South Carolina; Statesboro and Jesup, Georgia; and Jacksonville, Ocala, Zephyrhills, and Sarasota, Florida.

US 301 and SR 73 continue north from Statesboro through Sylvania to the Savannah River and the border with South Carolina.

Other concurrencies include U.S. 521 in Manning, U.S. 52 between Effingham and just south of Florence, U.S. 76 eastbound from east of Florence until Pee Dee, and finally joins U.S. 501 in Latta, where they both approach the South of the Border roadside attraction complex at the interchange with I-95 on the North Carolina-South Carolina border.

Entering Virginia just south of the community of Skippers, the route is paralleled by I-95 though much of the Southside region of Virginia, often times serving as the frontage road for I-95, deviating from it only in the city of Emporia, where it takes a more direct route through the center of the city.

In the Richmond metro area, it passes through the historic centers of the cities of Petersburg (where it meets its parent route, U.S. 1) and Colonial Heights.

Passing east of Chester, the two conjoined routes enter Richmond and pass through the Manchester portion of the city along a major boulevard, crossing the James River along the Robert E. Lee Memorial Bridge over Belle Isle Park, and following Belvidere Street and Chamberlayne Avenue through central Richmond.

At the extreme northern border of Richmond, the two routes split again, with U.S. 301 veering to the northeast past the community of Mechanicsville, Virginia and turning back to the north.

Traveling through numerous rural areas and small communities of central Virginia, the route turns northeast along a bypass around Bowling Green and passing close to Fort Walker as it turns to a more east-northeasterly direction, leaving the state in Dahlgren on the Harry W. Nice Memorial Bridge across the Potomac River.

U.S. Route 301 enters Maryland along the Governor Harry W. Nice Memorial Bridge across the Potomac River from Virginia.

A four-lane highway through most of the state, it is known locally as the Crain Highway and it connects several rural communities and small exurbs at the outer edges of the Washington metropolitan area, connecting communities such as La Plata, St. Charles, Waldorf and Upper Marlboro.

The freeway continues onto Maryland's Eastern Shore via the Chesapeake Bay Bridge, passing across Kent Island.

The four lane divided road continues generally northeast through the rural Eastern Shore region before crossing into Delaware and becoming a freeway.

Thus US 301 initially ran from US 17 (now US 76) at Pee Dee, South Carolina northeast through Dillon, Lumberton, Fayetteville, Dunn, Smithfield, Wilson, Rocky Mount, and Emporia, ending at US 1 in Petersburg, Virginia.

US 301 northbound turned east at Middletown along DE 299, then running north on US 13 to its end at Farnhurst at I-295 - the Delaware Memorial Bridge approach.

In the early 1970s, the northbound alignment was modified, continuing north on Middletown with DE 71 across the Chesapeake and Delaware Canal on the Summit Bridge.

After crossing the canal it continued north on DE 896 to Glasgow, where it turned east with US 40 to its merge with US 13 at State Road.

Signage currently now indicates that US 301 ends there,[citation needed] but on November 14, 2006, the Delaware Department of Transportation (DelDOT) announced that a new four-lane US 301 bypass will be built.

The bypass, which had been proposed since the 1950s, would go west of Middletown, alleviating traffic conditions in state's fourth most populous town, and then travel in a northeasterly direction, intersecting the current DE 896 near Boyds Corner and then terminate at DE 1 near the highway's Biddles Corner toll plaza.

When built, the road, like Delaware Route 1 and I-95, will charge a toll to cover the costs of building the new bypass, which is heavily used by trucks between Philadelphia and the Washington, D.C. metro areas.