The Gopher Frog (Rana capito) is a threatened species native to the southeastern longleaf pine ecosystem. Although once much more widespread across the southeastern United States, they now occur in North Carolina at only a handful of disjunct sites in the Coastal Plain and Sandhills regions of the state. The long-term persistence of these populations is thus a concern, as is a loss of genetic variation over time. We used mitochondrial and microsatellite markers to better understand the spatial structure of genetic variation and levels of genetic variability across these remaining populations in order to inform conservation and management decisions. Eight unique mitochondrial haplotypes were found, but these were all genetically similar to one another. Levels of genetic diversity based on the microsatellite analyses were similar across populations, but inbreeding coefficients in two populations were significant, suggesting a potential vulnerability to inbreeding depression. All disjunct populations showed significant genetic differentiation, which was not related to geographic distance. Conversely, within populations, the genetic relatedness of individuals between ponds decreased as distance between ponds increased. This kinship pattern is likely driven by strong breeding philopatry (individuals returning to the same ponds across years) and indicates that conservation actions at the scale of <1 km would primarily affect kin groups of Gopher Frogs, whereas conservation actions at scales ‡1.5 km would be needed to capture more distantly related individuals. Management efforts should thus focus on local metapopulation dynamics by maintaining multiple breeding ponds at each location, and by enhancing connectivity between these breeding ponds.