We assessed the effects of range disjunction, migratory habit, coloniality, and habitat structure on the genetic differentiation of North American Burrowing Owl (Athene cunicularia) populations. Burrowing Owls in North America comprise two forms or subspecies: A. c. floridana in Florida, separated by ∼1,500 km from the western form, A. c. hypugaea, which ranges from Texas to California and north to southern Canada. Burrowing Owls tend to be loosely colonial, and both the Florida populations and southerly populations of A. c. hypugaea from California to Texas are nonmigratory. To assess genetic structure, we examined 201 individuals from nine western and six Florida populations at seven highly variable microsatellite DNA loci. Mean gene diversity (H_exp) was higher in the west than in Florida (0.539 and 0.341, respectively; P < 0.05). Populations within subspecies were essentially panmictic (A. c. floridana: θ = 0.038, ρ = 0.014; A. c. hypugaea: θ = 0.014, ρ = 0.009) and even genetic differentiation across subspecies was modest (θ = 0.051, ρ = 0.014). Nevertheless, the western and Florida forms were easily distinguished by any of several criteria, such as allelic absences in Florida, assignment tests, and well-supported branches on the inferred phylogenetic tree. Genetic differentiation was at least twice as great in resident Florida (θ = 0.038) and California (θ = 0.021) populations as in migratory western populations (θ = 0.012), though 95% confidence intervals of theta estimates overlapped. We found no evidence of a genetic bottleneck that would result in evolutionary disequilibrium within subspecies. In the west, high observed heterozygosity values and evidence of gene flow suggest that population declines and patchy habitat, which currently imperil this species throughout much of its range, have not led to inbreeding or biologically meaningful genetic differentiation among the sampled populations.