Most of the original eastern and western portions of the route through roughly follows the path of the Memphis and Charleston Railroad, a railroad that predated the American Civil War and now operated by Norfolk Southern Railway as their Memphis-Chattanooga, Tennessee mainline.

The route follows rolling hills across the extreme northern part of the state, passing through Walnut and Corinth as it heads east.

Farther east, the route enters the Tennessee Valley and heads through Iuka before crossing into Alabama.

All of US 72 in Mississippi has been widened to four-lane highway, with the last two lane section between MS 302 and the Tennessee state line being completed in 2019.

The route parallels the Tennessee River east to Muscle Shoals, where US 72 Alternate splits from US 72.

The mainline turns north, passing through Muscle Shoals and crossing the Tennessee River, entering Florence on the opposite bank.

I-565/US 72 Alt., part of Corridor V of the Appalachian Development Highway System, terminates at the interchange while US 72 takes over the freeway alignment, joining Corridor V. I-565 is mentioned as the eastbound route and SR 20 is mentioned as the westbound route.

Just west of Moores Mill Road in Huntsville, US 72 becomes limited access over Chapman Mountain.

The stretch of US 72 paralleling I-59 to the far northeast in between Scottsboro and South Pittsburg contains multiple freeway-style Interchanges.

The route then follows a cut in the Cumberland Plateau made by the Tennessee River to the western outskirts of Chattanooga, where it interchanges with I-24 once more.

During the mid-1980s until the early 2000s, U.S. 72's westbound route in midtown Memphis was changed to switch from Poplar Avenue to a westbound off-ramp connection with Union Avenue (just as its name changed from Walnut Grove Road), instead of following Poplar west to East Parkway North.

In Tishomingo County, US 72 originally followed the current Road 172 / Quitman Street through Iuka before being bypassed in the mid-1980s.

The continuation of this segment west of Wolf River to Mississippi Highway 5 is signed Wyatt Road.

The possible former alignment then continues from the staggered junction heading west as a partially paved road.

[8] The original route once passed through Tuscumbia on what is named today the Old Lee Highway and Main Street.

Its route was later moved two blocks south through downtown Florence, from Tennessee Street to Dr. Hicks Boulevard.

East of Florence the original route is now County Highway 66 through downtown Rogersville and Killen.

Athens' Clinton and Washington Streets hosted a Business 72 route in earlier years.

US 72 Alternate between Decatur and Huntsville is part of Corridor V of the Appalachian Development Highway System.