Championed by Oklahoman Cyrus Avery in 1923 when the first talks about a national highway system began, US 66 was first signed in 1927 as one of the original U.S. Highways, although it was not completely paved until 1938.

    Avery was adamant that the highway have a round number and had proposed number 60 to identify it. A controversy erupted over the number 60, largely from delegates from Kentucky which wanted a Virginia Beach–Los Angeles highway to be US 60 and US 62 between Chicago and Springfield, Missouri. Arguments and counter-arguments continued and the final conclusion was to have US 60 run between Virginia Beach, Virginia and Springfield, Missouri, and the Chicago–Los Angeles, California route be US 62. Avery settled on "66" (which was unassigned) because he thought the double-digit number would be easy to remember as well as pleasant to say and hear.

    After the new federal highway system was officially created, Avery called for the establishment of the U.S. Highway 66 Association to promote the complete paving of the highway from end to end and to promote travel down the highway. In 1927, in Tulsa, Oklahoma, the association was officially established with John T. Woodruff of Springfield, Missouri elected the first president. In 1928, the association made its first attempt at publicity, the "Bunion Derby", a footrace from Los Angeles to New York City, of which the path from Los Angeles to Chicago would be on Route 66. The publicity worked: several dignitaries, including Will Rogers, greeted the runners at certain points on the route. The association went on to serve as a voice for businesses along the highway until it disbanded in 1976.

    Traffic grew on the highway due to the geography through which it passed. Much of the highway was essentially flat and this made the highway a popular truck route. The Dust Bowl of the 1930s saw many farming families (mainly from Oklahoma, Kansas, and Texas) heading west for agricultural jobs in California. Route 66 became the main road of travel for these people, often derogatorily called "Okies". And during the Depression, it gave some relief to communities located on the highway. The route passed through numerous small towns, and with the growing traffic on the highway, helped create the rise of mom-and-pop businesses (mainly as service stations, restaurants, and motor courts) up and down the highway.

    Much of the early highway, like all the other early highways, was gravel or graded dirt. Due to the efforts of the US Highway 66 Association, Route 66 became the first highway completely paved in 1938. Several places were dangerous, more than one part of the highway was nicknamed "Bloody 66" and gradually work was done to realign these segments to remove dangerous curves. However, one section (through the Black Mountains of Arizona) was fraught with sharp hairpin turns and was the steepest along the entire route—so much so that some early travelers, too frightened at the prospect of driving such a potentially dangerous road, hired locals to navigate the winding grade. The section remained until 1953—despite this, Route 66 continued to be a popular route.

    During World War II, more migration west occurred because of war-related industries in California. Route 66, already popular and fully paved, became one of the main routes and also served for moving military equipment. Fort Leonard Wood in Missouri was located near the highway, which was locally upgraded quickly to a divided highway to help with military traffic.

    In the 1950s, Route 66 became the main highway for vacationers heading to Los Angeles. The road passed through the Painted Desert and near the Grand Canyon. Meteor Crater in Arizona was another popular stop. This sharp rise in tourism in turn gave rise to a burgeoning trade in all manner of roadside attractions including teepee-shaped motels, frozen custard stands, Indian curio shops, and reptile farms. Meramec Caverns near St. Louis began advertising on barns, billing itself as the "Jesse James hideout". The Big Texan advertised a free 72 ounce steak dinner to anyone who could eat the whole thing in an hour. It also marked the birth of the fast-food industry: Red's Giant Hamburgs in Springfield, Missouri, site of the first drive-through restaurant, and the first McDonald's in San Bernardino, California. Changes like these to the landscape further cemented 66's reputation as a near-perfect microcosm of the culture of America, now linked by the automobile..