Birthplace and rise of US 66 The numerical designation 66 was assigned to the Chicago-to-Los Angeles route on April 30, 1926,^[in Springfield, Missouri. A placard in Park Central Square was dedicated to the city by the Route 66 Association of Missouri, and traces of the "Mother Road" are still visible in downtown Springfield along Kearney Street, Glenstone Avenue, College, and St. Louis streets and on Route 266 to Halltown, Missouri.

Championed by Avery when the first talks about a national highway system began, US 66 was first signed into law 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 who wanted a Virginia Beach–Los Angeles highway to be US 60 and US 62 between Chicago and Springfield, Missouri. Arguments and counterarguments continued throughout February, including a proposal to split the proposed route through Kentucky into Route 60 North (to Chicago) and Route 60 South (to Newport News). The final conclusion was to have US 60 run between Virginia Beach, Virginia, and Springfield, Missouri, and the Chicago–L.A. route be US 62.^[Avery and highway engineer John Page settled on "66", which was unassigned, despite the fact that in its entirety, US 66 was north of US 60.^[

The state of Missouri released its 1926 state highway map with the highway labeled as US 60.

After the new federal highway system was officially created, Cyrus Avery called for the establishment of the 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, 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 US 66.^[The publicity worked: several dignitaries, including Will Rogers, greeted the runners at certain points on the route. The race ended in Madison Square Garden, where the $25,000 first prize (equal to $376,793 in 2020) was awarded to Andy Hartley Payne, a Cherokee runner from Oklahoma. The U.S. Highway 66 Association also placed its first advertisement in the July 16, 1932, issue of the Saturday Evening Post. The ad invited Americans to take US 66 to the 1932 Summer Olympics in Los Angeles. A U.S. Highway 66 Association office in Oklahoma received hundreds of requests for information after the ad was published. The association went on to serve as a voice for businesses along the highway until it disbanded in 1976.

Traffic grew on the highway because of 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, Arkansas, Kansas, and Texas, heading west for agricultural jobs in California. US 66 became the main road of travel for these people, often derogatorily called "Okies" or "Arkies". 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, such as service stations, restaurants, and motor courts, all readily accessible to passing motorists.

The Chain of Rocks Bridge across the Mississippi River was built to carry the growing traffic of US 66 around the city of St. Louis

Restored Magnolia gasoline station museum on Route 66 in Shamrock in Wheeler County, Texas

Much of the early highway, like all the other early highways, was gravel or graded dirt. Due to the efforts of the U.S. Highway 66 Association, US 66 became the first highway to be 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 outside Oatman, Arizona, was fraught with 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 as US 66 until 1953 and is still open to traffic today as the Oatman Highway. Despite such hazards in some areas, US 66 continued to be a popular route. Notable buildings include the art deco–styled U-Drop Inn, constructed in 1936 in Shamrock, in Wheeler County east of Amarillo, Texas, listed on the National Register of Historic Places.^[24][25] A restored Magnolia fuel station is also located in Shamrock as well as Vega, in Oldham County, west of Amarillo.^[26]

During World War II, more migration west occurred because of war-related industries in California. US 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. When Richard Feynman was working on the Manhattan Project at Los Alamos, he used to travel nearly 100 miles (160 km) to visit his wife, who was dying of tuberculosis, in a sanatorium located on US 66 in Albuquerque.

In the 1950s, US 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 increase 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 (2.0 kg) steak dinner to anyone who could consume the entire meal in one hour. It also marked the birth of the fast-food industry: Red's Giant Hamburg 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.