Like many fragmented reptile populations, the ornate box turtle (Terrapene ornata ornata) is located in isolated, often small, populations in eastern Iowa. If populations are to remain viable, genetic diversity within these populations must be maintained, which is done most efficiently by migration among populations. Population viability was accessed using 3 microsatellite loci to determine population genetic structure in 2 locally dispersed subpopulations of ornate box turtles. Although these subpopulations were determined to be 1 large population with the normal range of heterozygosity, further examination revealed evidence of genetic divergence from a once larger population that is now geographically separated into fragments. We concluded that the central population studied was genetically healthy, and with proper management that continues to promote gene flow, the population should remain viable in the near future.