The black-footed ferret (Mustela nigripes) went extinct in the wild when the last 18 known ferrets were captured for a captive-breeding program. Following the success of the captive-breeding program, 146 genetically nonessential ferrets were released at the Conata Basin, South Dakota, from 1996 to 1999. We conducted a genetic analysis of the Conata Basin black-footed ferret population from 2001 to 2003 to determine if genetic variation had been lost since the cessation of reintroductions and if demographic- and genetic-based estimates of effective population size (N_e) accurately predicted observed levels of heterozygosity. We used DNA from wild-born kits (n  =  254) in the Conata Basin population (2001–2003) to calculate current genetic diversity levels. Both allelic diversity (A  =  2, both subpopulations) and mean heterozygosity were low for both subpopulations—0.39 ± 0.12 SE in Agate-Sage Creek and 0.39 ± 0.16 SE in Heck Table—but not significantly different from estimates made in 1999. We found no significant difference between observed and expected heterozygosity levels. Demographic-based estimates of N_e were an order of magnitude higher than genetic-based estimates of N_e, but the 2 estimates provide a range of N_e values for the population. This study shows that the Conata Basin ferret population is able to maintain its genetic diversity over time despite its population history.