In Missouri, the highway ran from downtown St. Louis at the Mississippi River to the Kansas state line west of Joplin.

By the early-to-mid-19th century, settlers laid a telegraph line along the road (it continued south from Springfield to Fort Smith, Arkansas).

In this city, US 66 was concurrent with US 65 for several miles, and also served as the original western terminus of US 60, which does not intersect with US 66 in Missouri anymore.

At Rolla, about halfway from Springfield to St. Louis, US 66 intersected with US 63 (Bishop Avenue) and passed by the Missouri University of Science and Technology.

The older highway passes through downtown Pacific in a scenic area under steep bluffs.

Bypass US 66 was routed over the Mississippi on the north side of St. Louis via the Chain of Rocks Bridge.

[8] The Pulaski County Courthouse in Waynesville built 1903 in Romanesque Revival style now houses a museum.

[10] The Red Cedar Inn, a log cabin restaurant opened in 1933 (and closed in 2005), was built by James and Bill Smith in Pacific.

[11] The Big Chief Restaurant in Wildwood opened in 1928 as the Big Chief Hotel, a roadside tourist cabin court with 62 rooms (each constructed as an individual cabin with its own garage) and an on-site restaurant, dance hall, Conoco filling station, playground and general store.

The cabins, which served as housing for Weldon Spring Ordnance Works workers during World War II, were later demolished; the restaurant, closed in 1949, was restored in the 1990s and reopened.

Its restaurant is now a souvenir shop and its filling station is no longer operational, but it continues to offer lodging to US 66 travellers more than seventy-five years later.

Not yet listed on the National Register of Historic Places, the 1939 streamline moderne Boots Court originally touted "a radio in every room" long before the 1949 opening of the 66 Drive-In cinema and the 1953 sign-on of the first local Joplin-Springfield television stations.

The streamline moderne Coral Court Motel in Marlborough (an inner St. Louis suburb) was demolished in 1995, its 8.5 acre site sold for over a million dollars for suburban residential development, while John's Modern Cabins in Newburg, abandoned since the 1970s, have been allowed to slowly deteriorate beyond repair.

The Meramec River U.S. 66 Bridge - J421 was built in 1931 when US 66 was rerouted to pass through Times Beach, a working-class resort established in 1925 and abandoned in 1983 due to dioxin contamination.

During the summer of 2016 it was announced that the Solar Roadways firm in Sandpoint, ID will be attempting trials of its smart highway technology in and around the community of Conway, MO, as sponsored by MoDOT, at Conway's Route 66 Welcome Center and Museum, as sidewalk paving.

Eventually, if the trials go well, the firm's hexagonal roadway panels would even be used as paving for sections of the historic Route 66 roadbed itself in time.