Genetic variability and structure of nine black-tailed prairie dog (BTPD, Cynomys ludovicianus) colonies were estimated with 15 unlinked microsatellite markers. A plague epizootic occurred between the first and second years of sampling and our study colonies were nearly extirpated with the exception of three colonies in which prairie dog burrows were previously dusted with an insecticide, deltamethrin, used to control fleas (vectors of the causative agent of plague, Yersinia pestis). This situation provided context to compare genetic variability and structure among dusted and non-dusted colonies pre-epizootic, and among the three dusted colonies pre- and post-epizootic. We found no statistical difference in population genetic structures between dusted and non-dusted colonies pre-epizootic. On dusted colonies, gene flow and recent migration rates increased from the first (pre-epizootic) year to the second (post-epizootic) year which suggested dusted colonies were acting as refugia for prairie dogs from surrounding colonies impacted by plague. Indeed, in the dusted colonies, estimated densities of adult prairie dogs (including dispersers), but not juveniles (non-dispersers), increased from the first year to the second year. In addition to preserving BTPDs and many species that depend on them, protecting colonies with deltamethrin or a plague vaccine could be an effective method to preserve genetic variability of prairie dogs.