The Gray Fossil Site is a rich Hemphillian (North American Land Mammal Age) locality in northeastern Tennessee, USA, and has produced tens of thousands of fossils of multiple taxa including hundreds of individual snake skeletal remains. One of only five sites of similar age east of the Mississippi River, the Gray Fossil Site is crucial to our understanding of snake evolution and diversity. Microfauna were collected by screenwashing sediment and picking under a microscope. Specimens were identified using a suite of measurements and characters. Analyzed here are vertebral fossils identified as belonging to various colubrid taxa including Worm Snakes (Carphophis sp.), Ring-Necked Snakes (Diadophis sp.), Hook-Nosed Snakes (Gyalopion sp.), and Hognose Snakes (Heterodon meadi sp. nov.). Carphophis sp. and Gyalopion sp. represent first occurrences in the Mio-Pliocene and dramatically extend their temporal range from the Pleistocene. The four snakes, including one new species, are in accordance with the previously identified taxa by Jasinski and Moscato (2017; Journal of Herpetology 5:245–257), represent a modern assemblage of advanced snakes by the Mio-Pliocene and support a forested environment in northeastern Tennessee.