Understanding species distributions is important for determining whether conservation efforts are necessary. In central Kentucky, an area of potential range overlap occurs between northern leopard frogs (Lithobates pipiens), which is a species of special concern, and southern leopard frogs (L. sphenocephalus). Their distributions are not completely delineated because of their low abundance in central Kentucky and similarity in morphology. Thus, use of genetic markers is necessary for identification. We surveyed for new, and sampled known, populations with a focus on the area of overlap in central Kentucky. We sequenced a portion of the mitochondrial 16S rRNA gene for 55 individuals from 16 populations. Maximum parsimony analysis separated individuals into two completely unresolved lineages representing the two species. Species were distributed as predicted, and no areas of fine-scale overlap were detected, although we did find a population of southern leopard frogs 10 km north-northwest of a northern leopard frog population. It was difficult to find L. pipiens at historically recorded populations, but we documented them in two counties where identification was previously unknown because morphology was intermediate between the species. Although genetic data reliably distinguished the species, they were inconclusive about whether hybridization occurs; future work is necessary to address this question.