Barriers to dispersal often cause evolutionary divergence. In the present, identification of geographic boundaries between lineages can indicate the historic presence of biogeographic barriers. In eastern North America, these patterns often arose during periods when changes in climate accompanied the origin of topographic features that hinder gene flow through expansion of river systems, changes in sea level, and restriction of organisms to refugia. However, organisms’ responses to changing landscapes is often complex. Not all taxa, even those with similar ecologies, exhibit effects on gene flow from the same changes in geographic features and climate; there is often nuance in these evolutionary shifts. In this study, I use the banded water snake, Nerodia fasciata, as a model to test for phylogeographic structure associated with potential biogeographic barriers. While this species is found throughout the U.S. southeastern coastal plain region, previous phylogeographic research has been limited to Florida, a deficiency I address here. I sequenced the mitochondrial gene cytochrome b (cytb) and the nuclear gene prolactin receptor (PRLR) for phylogenetic analysis across the range of N. fasciata. Additionally, biogeographic modeling was used to investigate the historic distributions of this group. Results indicate that this species first diversified during the late Miocene and early Pliocene and may have historically been isolated to a glacial refugium in the western part of its range and to the Florida peninsula by high sea levels. These findings are consistent with patterns observed in certain other snake species distributed throughout the American southeast. Phylogeographic breaks were not found at major river systems, while ranges of the subspecies of this group better fit the distributions of its major mitochondrial lineages. This research contributes new information on the systematics of N. fasciata and helps to explore how organisms respond to changing geography across continental land masses, providing a model for testing southeastern biogeography in other taxa.