The Trans-Mexican Volcanic Belt and surrounding areas contain substantial biological diversity. The mountains that make up the Trans-Mexican Volcanic Belt are a hypothesized biogeographic barrier for the terrestrial fauna found in the region. Several phylogeographic studies have provided genetic evidence in support of this historical narrative; however, the species examined represent a small percentage of the diversity found in this part of Mexico. Thus, additional studies are needed to identify concordant phylogeographic patterns and infer the historic species composition of particular ecoregions. In this study we investigated genetic variation in the Lowland Burrowing Treefrog, Smilisca fodiens, a species that occurs on both sides of the Trans-Mexican Volcanic Belt. We used mitochondrial (12S and 16S ribosomal subunits; 1039 base pairs [bp]) and nuclear (tyrosinase precursor; 513 bp) DNA to perform phylogenetic analyses on frogs from several localities in Mexico. Mitochondrial DNA supported two well-defined clades that correspond to populations found north and south of the Trans-Mexican Volcanic Belt, respectively. These analyses of matrilineal lineages also found higher levels of genetic diversity south of the Trans-Mexican Volcanic Belt. Although our nuclear DNA analysis did not reveal a phylogeographic split at the Trans-Mexican Volcanic Belt, we observed higher genetic variation among our southern samples, similar to the mitochondrial analyses. Our results are consistent with studies in other sympatric taxa that propose the Trans-Mexican Volcanic Belt as a biogeographic barrier. Additionally, our results suggest recent northern range expansion of S. fodiens. We suggest retaining S. fodiens as a single species until future work can clarify the amount and direction of gene flow between the mitochondrial clades.