We investigated genetic variability in 101 individuals of the Cagle's Map Turtle (Graptemys caglei) collected from across most of its extant distribution in the Guadalupe River and from a site on the San Marcos River of Texas. Analyses of allelic variation in microsatellite loci indicated individuals from the San Marcos River were genetically similar to conspecifics from the middle Guadalupe River. Turtles from the upper Guadalupe River, on the other hand, were genetically divergent from middle Guadalupe River individuals. Isolation by distance appears to play a major role in genetic structuring within the Guadalupe River. Anthropogenic features (e.g., dams and reservoirs), while they may be expected to restrict gene flow, appear to have had little impact on genetic structure.