The three-lined salamander, Eurycea guttolineata, is a wide-ranging lungless salamander in the southeastern United States. We have sampled seventy-seven populations spanning the entire distribution of the three-lined salamander. We have amplified, purified, and sequenced 872 base pairs of the mitochondrial gene Cty-b and used this data to reconstruct the evolutionary history of three-lined salamanders. Our Bayesian phylogenetic reconstruction reveals a pattern of extreme genetic homogeneity across the range of three-line salamanders. However, in the Florida Parishes of Louisiana, we found that populations are highly differentiated genetically from adjacent areas. Here we examine potential biogeographic barriers that may explain this pattern of differentiation.