Burrowing Owls (Athene cunicularia) have a large geographic range spanning both North and South America and resident populations occur on many islands in the eastern Pacific Ocean and the Caribbean Sea. Many owl populations are isolated and disjunct from other populations, but studies on genetic variation within and among populations are limited. We characterized DNA microsatellite variation in populations varying in size and geographic isolation in the Florida (A. c. floridana), the Western (A. c. hypugaea), and the Clarion (A. c. rostrata) subspecies of the Burrowing Owl. We also characterized genetic variation in a geographically isolated population of the western subspecies in central Mexico (near Texcoco Lake). Clarion Burrowing Owls had no intrapopulation variation (i.e., fixation) at 5 out of 11 microsatellite loci, a likely outcome of genetic drift in an isolated and small population. The Florida subspecies had only polymorphic loci but had reduced levels of genetic variation compared with the more-widespread western subspecies that occurs throughout western North America. Despite the extensive geographic distribution of the Western Burrowing Owl, we found genetic differentiation between the panmictic population north of the Trans-Mexican Volcanic Belt and the resident Texcoco Lake population in central Mexico.