Editor Anthony Pinker was amused by the notes he got from the network for this episode. Despite all the multiple deaths and violence in the show, Pinker was expressly told not to depict Cassie on top of Dean in their sex scene. True to the rebellious nature of the Supernatural cast and crew, they did exactly that.
Athens Ohio, as mentioned in the beginning of the episode, is regarded as one of the most haunted areas in the U.S.A.
The setting of the fire to the church in 1963 that killed the children inside during their choir practice may be an allusion to the 16th Street Baptist Church bombing that occurred in 1963 in Birmingham, Alabama, killing 4 of the 5 children inside during choir practice.
The episode title referencing the 1960's TV series Route 66 (1960) is especially apt because that show established the format used by many later shows, including 'Supernatural'. A man, or two men (or two women in the case of Xena: Warrior Princess (1995)), with no fixed home, no regular jobs, wander the countryside helping out folks who are in trouble. These shows have few if any sets and are mostly filmed on location.
Due to licensing issues, many songs in season 1 were replaced, thus the song that plays at the end of this episode is "Line of Love" by The Minors. Originally, it was "Can't Find My Way Home" by Blind Faith, which coincidentally was used again in the season 9 finale Do You Believe in Miracles (2014).