The San Marcos salamander (Eurycea nana), endemic to the headwaters of the San Marcos River, is federally threatened because of its restricted range and threats to its habitat. Captive propagation efforts for E. nana were initiated in 1996 to maintain a captive population for restocking in the event that E. nana becomes extinct. We surveyed DNA sequence data for a mitochondrial and a nuclear gene region (ND4 and rag1) and 281 AFLP markers to obtain baseline data on the level of genetic diversity across the known range of E. nana and to test for population subdivision within its restricted range. Next, we compared levels of genetic variation between E. nana in the wild and captive-born individuals from the captive population to assess the efficacy of the captive breeding program. We investigated inbreeding in the captive population by testing for deviations from Hardy-Weinberg equilibrium at the rag1 locus for captive-born individuals. The level of genetic diversity detected in wild E. nana was similar to that of other Eurycea populations in central Texas, and we found no evidence of population structure across the range of E. nana. The surveyed captive-born E. nana contained reduced genetic diversity at some loci, but similar genetic diversity at others relative to the wild, and there was no evidence of inbreeding within the wild-caught individuals that sired these captive-born individuals. The data we have obtained regarding the level of genetic variation in wild E. nana and in the captive population will provide baseline information for this species.