Canis latrans (Coyotes) are a management concern in the southeastern US because of their potential impacts on agriculture, other wildlife species, and human health and safety. This region is part of a recent range expansion by Coyotes, and information about their population structure in the southeastern US is lacking. In this study, we used microsatellite DNA to assess genetic diversity and population structure among Coyotes in east-central Alabama. We detected high genetic diversity (H_E = 0.78) and no population structure across the total sampling area. Additionally, we investigated population structure between urban and rural groups. We detected low but significant population structure between these groups, which may be biologically meaningful. We discuss the implications of this result in the context of potential management strategies. Overall, our study sought to provide information about the molecular ecology of Coyotes within a region of recent range expansion.