Reintroduction of game species and augmentation of declining populations were carried out in many areas of Europe during the last century. In some cases, such actions have threatened the diversity of remnant endemic taxa, thus raising concerns for their conservation. The Italian peninsula hosts relict populations of native roe deer, which include the subspecies Capreolus capreolus italicus. Recent genetic investigations and historical information suggest that roe deer populations in Italy have different origins (native, introduced, and admixed). Here, we conducted genetic analyses in 3 areas of central Italy (provinces of Arezzo, Pisa, and Parma), each characterized by subareas containing roe deer with different genetic ancestries and zones of contact among them. We analyzed mitochondrial (control region) and nuclear (microsatellites) data for a total of 304 individuals. In all 3 study areas, both marker systems revealed a decreasing frequency of the C. c. italicus lineage from south to north. We found the highest degree of admixture in contact zones, but local patterns varied on the basis of population history and landscape features. Notably, we found relatively high genetic differentiation between the extremes of each sampling area, despite the limited spatial scale studied and time elapsed since introductions. Further, our analyses of a population in the Apennine Mountains reveal that this region may maintain previously undetected native diversity. Finally, the correspondence between data from mitochondrial and nuclear genomes is consistent with the absence of sex-biased dispersal in roe deer. Further investigations are needed to elucidate which conditions may favor the persistence of native diversity.