Small, isolated populations are vulnerable to loss of genetic diversity through inbreeding and genetic drift. Sylvatic plague due to infection by the bacterium Yersinia pestis caused an epizootic in the early 1990s resulting in declines and extirpations of many black-tailed prairie dog (Cynomys ludovicianus) colonies in north-central Montana, USA. Plague-induced population bottlenecks may contribute to significant reductions in genetic variability. In contrast, gene flow maintains genetic variability within colonies. We investigated the impacts of the plague epizootic and distance to nearest colony on levels of genetic variability in six prairie dog colonies sampled between June 1999 and July 2001 using 24 variable randomly amplified polymorphic DNA (RAPD) markers. Number of effective alleles per locus (n_e) and gene diversity (h) were significantly decreased in the three colonies affected by plague that were recovering from the resulting bottlenecks compared with the three colonies that did not experience plague. Genetic variability was not significantly affected by geographic distance between colonies. The majority of variance in gene frequencies was found within prairie dog colonies. Conservation of genetic variability in black-tailed prairie dogs will require the preservation of both large and small colony complexes and the gene flow among them.