As gopher tortoises (Gopherus polyphemus) increasingly become threatened throughout their range in Florida, USA, the need for management and conservation will intensify. Here we evaluate the forensic applicability of genetic assignment tests based on microsatellite genotypic data to 1) accurately assign individuals in our genetic database to the sample location or population of origin and 2) determine the origin of 6 confiscated tortoises. Overall, we could correctly assign 90% of the individuals in the database to their population of origin, but we were unable to determine the source of the confiscated tortoises. However, these individuals are unlikely to have come from any of our sampled sites and all 6 may have come from the same population. This approach can be used by law enforcement personnel to identify the origin of confiscated tortoises as well as by developers and wildlife managers to determine the genetic appropriateness of potential recipient populations when it is necessary to relocate individuals.