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We examined the genetic structure of 5 Wyoming cougar (Puma concolor) populations surrounding the Wyoming Basin, as well as a population from southwestern Colorado. When using 9 microsatellite DNA loci, observed heterozygosity was similar among populations (HO = 0.49–0.59) and intermediate to that of other large carnivores. Estimates of genetic structure (FST = 0.028, RST = 0.029) and number of migrants per generation (Nm) suggested high gene flow. Nm was lowest between distant populations and highest among adjacent populations. Examination of these data, plus Mantel test results of genetic versus geographic distance (P ≤ 0.01), suggested both isolation by distance and an effect of habitat matrix. Bayesian assignment to population based on individual genotypes showed that cougars in this region were best described as a single panmictic population. Total effective population size for cougars in this region ranged from 1,797 to 4,532 depending on mutation model and analytical method used. Based on measures of gene flow, extinction risk in the near future appears low. We found no support for the existence of metapopulation structure among cougars in this region.