The Gray Fossil Site (GFS) of northeastern Tennessee is a late Hemphillian fossil locality in the southern Appalachian mountain region of eastern North America with a diverse vertebrate fauna. Snakes make up a substantial microfossil portion of the GFS herpetofauna, particularly the Colubridae, comprised of members of the Colubrinae and Natricinae. Seven colubrid taxa have been identified from the site so far, including three natricines (cf. Neonatrix, Nerodia, Thamnophis) and at least four colubrines (Coluber/Masticophis, Pantherophis, Pituophis, gen. et sp. nov.). Indeed, cf. Neonatrix and the new genus (and species) are the only extinct genera identified. Although Neonatrix is tentatively identified for the first time east of Nebraska, the new species represents a distinct taxon. In addition, the oldest reported definitive occurrence of Masticophis is presented herein. Some of the snakes suggest a pond or other aquatic habitat at GFS, particularly cf. Neonatrix and Nerodia, whereas others, such as Masticophis and Pituophis, tend to prefer more open forested habitats. The GFS represents a poorly understood region of North America at a crucial time period in snake evolution, and its study may help us further understand the modern snake fauna present today in midcontinental and eastern North America.