U.S. 65 begins in Clayton, Louisiana and proceeds northward to Waterproof, St. Joseph, and Newellton, all in Tensas Parish.

It is designated as part of Arkansas' Great River Road from this point north through Lake Village, McGehee, and Dumas.

US 65 was later relocated to a bypass corridor on the north side of Pine Bluff, dubbed the Downtown Expressway.

As US 65 progressed into North Little Rock's Levy neighborhood, its alignment shifted east of the railroad along Pike Avenue, turning northwest along Parkway Drive to converge with its original route near the city's Amboy neighborhood.

Later, the Levy-to-Amboy segment was relocated again along the west side of the railroad via MacArthur Drive, eventually converging with its original route.

The highway was eventually relocated along Interstate 40, where it joins its original route on the north side of town via the city's Skyline Drive.

US 65 continues north through Greenbrier, Clinton, and Marshall before crossing the Buffalo River near Tyler Bend.

The four-lane expressway continues through Hollister and Branson toward the Springfield metro area.

North of Branson is an interchange with the Ozark Mountain Highroad (a freeway spur) and U.S. Route 160.

Just north of Route EE (Highlandville exit), US 65 returns to freeway status.

The highway enters the city proper, reaching downtown before returning to end at Interstate 35.

Its current endpoints were established in 1980 when a segment paralleling Interstate 35 was dropped in Minnesota.

In 1980, U.S. 65 in Minnesota merged with Interstate 35 until it separates in downtown Minneapolis leaving the city in the Central Avenue corridor.

Beginning at Washington Avenue in downtown Minneapolis, Minnesota Highway 65 travels north through Cambridge, Mora, and McGregor before terminating at an intersection with U.S. Highway 71 in Littlefork (just south of International Falls).

[7] Legally, the Mississippi section of U.S. 65 is defined in Mississippi Code Annotated § 65-3-3, as follows: "U.S. 65- Begins at the west end of the Mississippi River Bridge at Natchez, Adams County, and extends in an easterly direction to U.S. 61 and thence continues south jointly with U.S. 61 to the Mississippi-Louisiana state line south of Woodville, Wilkinson County."

Through central Arkansas, old 65 (now Highway 365) follows close to Interstate 40 between Conway and North Little Rock.

A realignment around 1931 which involved a more direct path from Palarm Creek to Conway resulted in the road being relocated from the West to the East side of the railroad.

As a result, the city of Mayflower Arkansas moved slightly as well to stay on the new route.