Using 8 polymorphic microsatellite loci, we explored genetic variability in Texas spiny softshell turtles (Apalone spinifera emoryi) in the region of Big Bend National Park (BBNP) and the Lower Rio Grande Valley (LRGV), which are located in western and southern regions, respectively, of the distribution of the subspecies in Texas. The presence of multiple anthropogenic stressors, such as river flow alterations, human population expansion, and direct harvest, motivated us to evaluate whether genetic consequences of these stressors have become apparent in this species. A low but significant level of genetic differentiation was detectable between these 2 regions. There was also detectable isolation by distance among the turtles in LRGV but not among turtles in BBNP, possibly because the LRGV localities were discontinuous ponds, whereas the BBNP localities were continuously joined stretches of the Rio Grande. We detected no evidence of a recent population bottleneck in BBNP or the LRGV. However, turtles are generally long-lived and, because harvest activity peaked in the 1990s, it is likely that detecting harvest-related changes would be challenging. Continuous long-term sampling is necessary to evaluate the genetic consequences of anthropogenic pressures.