Anthropogenic habitat fragmentation and reduction are major causes of population declines and extinction. As these processes intensify, our ability to rescue imperiled taxa is critically dependent on an understanding of historical, demographic, and genetic parameters of diminishing populations. We assessed the effects of recent geographic isolation and population reduction on genetic variability for endangered Dusky Gopher Frogs, Rana sevosa. Only two populations of R. sevosa exist, each is geographically isolated and restricted to a single breeding pond, and one of them may have gone locally extinct. Therefore, we studied the largest and perhaps only population of R. sevosa. The only option for comparison to non-isolated populations was of its ecologically similar sister species (other Gopher Frogs, R. capito) and of the sister species to R. sevosa and R. capito (Crawfish Frogs, R. areolata). Variation in seven microsatellite DNA loci was assessed for each population to determine the effects of isolation and population bottleneck on R. sevosa. In comparison to the average non-isolated population, R. sevosa had significantly lower genetic variation and a strong signature of population bottleneck. In fact, R. sevosa had H_O that was 72%, H_E that was 81%, and A that was 61% of the average non-isolated population. Results indicated a severe, negative genetic consequence of recent population reduction and geographic isolation via lack of gene flow, enhanced effects of drift, and inbreeding. Extensive demographic data have been collected for R. sevosa beginning when the species was rediscovered in 1987 and continuing through our study. These previously collected demographic data aid in interpretation of our genetic data and discussion of implications for conservation and management.