Habitat alteration, agricultural control, recreational shooting, and most recently, sylvatic plague (caused by Yersinia pestis) contributed to local extinctions and a steady decline of black-tailed prairie dog (Cynomys ludovicianus) throughout its range. As a consequence, prairie dogs currently live in metapopulations, where their overall persistence will depend on a balance between extinction of colonies and recolonization from extant colonies. Patterns of genetic similarity among colonies, as measured by neutral molecular markers, provide an estimate of the dispersal and gene flow among colonies within prairie dog metapopulations. We sampled 13 colonies of black-tailed prairie dogs in short-grass prairie of northern Colorado, 100-km east of Fort Collins, Colorado. We used historical records and genetic analysis to show that colonies undergo regular extinctions, which subsequently are recolonized by individuals from multiple source colonies. We examined 155 individuals for variation at 7 microsatellite loci and found moderate levels of genetic differentiation among colonies (Θ [=F_ST] = 0.118). We also used assignment and exclusion tests based on multilocus genotypes of individuals to determine the probability that individuals originated from the same colony in which they were captured. About 39% of individuals could not be assigned to colonies where they were captured, indicating they were either immigrants (adults) or the offspring of immigrants (adults and juveniles). We tested for genetic isolation by distance among colonies by comparing genetic distances to geographic distances between colonies. Akaike's Information Criterion for model selection revealed that dispersal most likely occurred along low-lying dry creek drainages connecting isolated colonies. Genetic distances between colonies were also related to ages of colonies; older colonies were more similar genetically than younger colonies. This underscores the importance of dispersal among prairie dog colonies and has important implications for persistence of prairie dog metapopulations, in which all colonies, regardless of size, are vulnerable to extinction from plague.