There is a general expectation that urban populations will be fragmented, and the movement of individuals will be restricted leading to low effective population sizes, low genetic diversity, higher inbreeding, and higher differentiation than populations living in more continuous habitat. Most studies have been conducted on birds and mammals living in large urban areas while very few studies have been conducted on the genetic structure of small reptiles living within the urban matrix. In this study, we compare the genetic diversity and differentiation of Texas horned lizards that are found in four small towns (Kenedy, Karnes City, Rockdale, Smithville) in Texas and at Tinker Air Force Base, Oklahoma to populations that occur in 16 natural areas and to an introduced population in South Carolina. We also present more detailed spatial genetic data and home range data for several of the towns. Texas horned lizards (ii) living in small towns have lower genetic diversity, higher differentiation, and smaller effective population sizes than populations located in more natural mainland areas. There was evidence for human-mediated movement of lizards into town, however it has not been enough to counteract the effects of drift. Home range size is smaller in town than more natural areas. Genetic patterns suggest dispersal occurs over short distances and is inhibited across areas with a high percent of impervious surface and major roads. These data suggest that effective planning to maintain suitable habitat and corridors to facilitate movement are critical to maintaining small terrestrial species like the Texas horned lizard and must be integrated into the early stages of urban development.