The illegal trade has been threatening tortoise populations worldwide for decades. Nowadays, however, DNA typing and forensic genetic approaches allow us to investigate the geographic origin of confiscated animals and to relocate them into the wild, providing that suitable molecular tools and reference data are available. Here we assess the suitability of a small panel of microsatellite markers to investigate patterns of illegal translocations and to assist forensic genetic applications in the endangered Mediterranean land tortoise Testudo hermanni hermanni. Specific allelic ladders were created for each locus and tested on several reference samples. We used the microsatellite panel to (i) increase our understanding of the population genetic structure in wild populations with new data from previously unsampled geographic areas (overall 461 wild individuals from 28 sampling sites); (ii) detect the presence of non-native individuals in wild populations; and (iii) identify the most likely geographic area of origin of 458 confiscated individuals hosted in Italian seizure and recovery centers. Our analysis initially identified six major genetic clusters corresponding to different geographic macro-areas along the Mediterranean range. Long-distance migrants among wild populations, due to translocations, were found and removed from the reference database. Assignment tests allowed us to allocate approximately 70% of confiscated individuals of unknown origin to one of the six Mediterranean macro-areas. Most of the assigned tortoises belonged to the genetic cluster corresponding to the area where the respective captivity center was located. However, we also found evidence of long-distance origins of confiscated individuals, especially in centers along the Adriatic coast and facing the Balkan regions, a well-known source of illegally traded individuals. Our results clearly show that the microsatellite panel and the reference dataset can play a beneficial role in reintroduction and repatriation projects when confiscated individuals need to be re-assigned to their respective macro-area of origin before release, and can assist future forensic genetic applications in detecting the illegal trade and possession of Testudo hermanni individuals.