Identifying cryptic biodiversity is fundamental to evolutionary biology and to conservation efforts. This study investigated range-wide genetic diversity of Gopher Frogs, Lithobates capito, across the southeastern United States coastal plain to determine implications for taxonomy and conservation. We collected data for two mtDNA regions in 21 populations to identify genetic structure across the geographic distribution of the species. Based on population genetic, phylogenetic, and genealogical analyses, we recovered three reciprocally monophyletic mtDNA lineages corresponding to mainland coastal plain populations and two lineages within peninsular Florida. Breakpoints for these lineages did not occur in previously identified hotspots of amphibian phylogeographic breaks and did not follow currently recognized subspecies designations. We recommend these lineages be recognized as separate distinct population segments and be considered separately by the U.S. Fish and Wildlife Service for listing under the Endangered Species Act. Additionally, we propose an evolutionary hotspot for amphibians that deserves further attention.