One memorable episode featured Boris Karloff, Lon Chaney, Jr., and Peter Lorre. Karloff and Chaney were given the chance to don their famous Universal Frankenstein Monster and Wolf Man make-up for the first time in a few decades.

Although the series was called "Route 66", many of the episodes were set in areas in the United States, through which Route 66 did not travel. For example, Route 66 begins in Chicago and ends in Los Angeles, or vice versa, but two episodes were shot in New England, one in Maine, season four,, episode two, "Same Picture, Different Frame", and one in Vermont, season four, episode three, "Come Out, Come Out, Wherever You Are". Both aired in the fall of 1963, and starred Martin Milner and Glenn Corbett.

According to Glenn Corbett, none of the episodes, in which he appeared, were shot along the real Route 66. When he asked the producers why, he was told that the scenery along the actual highway wasn't considered interesting enough.

Martin Milner and George Maharis were reunited in SST: Death Flight (1977).

Three future movie stars nearly became regulars on this series. When the show was first getting off the ground, a young unknown named Robert Redford auditioned for the role of Tod Stiles, but was beaten out by Martin Milner. When George Maharis left the show in late 1962, the producers searched for a replacement. Burt Reynolds was approached, but he was then involved on Gunsmoke (1955) playing Quint Asper. Another actor who wanted to do the series, was future Academy Award winner Robert Duvall, who was even auditioned in one episode. The producers felt that Duvall was not handsome enough, however, and went with Glenn Corbett instead.

George Maharis walked off the show during the third season during a bout with hepatitis, although it has been alleged that he was dissatisfied with his contract and wanted to work in film, and that he had on-going clashes with the show's producers. He was replaced by Glenn Corbett, but audiences did not like the change, and the show was cancelled as a result.

The Corvette was replaced every three thousand miles. Chevrolet was the show's sponsor. It was never explained how Tod was able to get a new Corvette so often.

The series' writers travelled around the U.S. looking for locations, about which to write episodes. Every episode was filmed on-location.

Martin Milner drove a 1962 Chevrolet Corvette during the show's run.

The original pilot of this series aired as an episode of Naked City (1958) titled "Four Sweet Corners". Maharis played Johnny Gary and Robert Morris played Link Ridgeway. Morris died before the series was picked up.

The Corvette was never red, as often believed. It was light blue in the first season, beige in the second season, and tan in the third season. The colors were chosen because they photographed well in black and white.

The show was originally to be called "The Searchers", which would have made a lot more sense, but producers feared there would be confusion with the very popular John Wayne western by that name that came out four years earlier.

The planned fifth season would have taken place in Europe. It would have been the first American series to be filmed overseas. But the series was cancelled after the fourth season. I Spy (1965) eventually became the first American series to do so.

Maharis became ill after filming the episode "Even Stones Have Eyes", in which he spent several hours in a freezing river. His illness worsened after filming "There I Am - There I Always Am", in which he spent several hours in the cold water off of Catalina Island. Maharis continued to work on the series, as he was not allowed to take a break from filming to recover. He eventually contracted hepatitis from an injection given to him by a doctor brought in by the studio, and had to take time off for his health. Maharis returned after a month, but suffered a relapse. He finally decided to leave the series, rather than risk his health any further. Executive Producer Herbert B. Leonard sued Maharis for breach-of-contract, which was settled out of court. Glenn Corbett was cast as Maharis' replacement, and Buz was never mentioned again.

Nat "King" Cole's song "(Get Your Kicks on) Route 66" was considered for the show's theme. But CBS wanted an original theme to avoid paying the expensive royalties for Cole's song. Nelson Riddle's "Route 66 Theme" hit number thirty on the Billboard Hot 100, and was nominated for two Grammys. George Maharis recorded a version of Cole's song as a tribute.

The series was designed as essentially an anthology that had the main two characters of the series in all the stories.

Author David Morrell was a fan of the series.