The Dinaric Mountains in Slovenia, Croatia, and Bosnia and Herzegovina provide a unique system to address the effects of past hunting on the genetic structure of northern chamois (Rupicapra rupicapra) and possible hybridization in the contact zone in the Velebit Mountains. The northern Dinaric Mountains should be occupied by alpine chamois (Rupicapra rupicapra rupicapra), whereas the central and southern areas are inhabited by the Balkan chamois (R. rupicapra balcanica). This is the first study to characterize the genetic variation in chamois populations in the area. We used microsatellite and mitochondrial markers to analyze the genetic variation and structure of chamois populations from different geographical areas with different histories. Specifically, we explored the influence of recent human translocations and geographical isolation on the genetic architecture of chamois populations in the assumed contact zone. We successfully genotyped 74 individual samples and the number of alleles/locus ranged from 6 to 20 with a mean of 9.20. Allelic richness across populations ranged from 2.94 in the Prenj Mountains, Bosnia and Herzegovina to 3.56 in the Biokovo Mountains, Croatia. A similar pattern was also observed for heterozygosity, ranging between 0.729 and 0.572, and expected heterozygosity, ranging between 0.762 and 0.644 in the Prenj and Biokovo mountains, respectively. The global genetic distance (FST) for 7 population samples was 0.103 ±0.047 (range = 0.0156–0.185). The STRUCTURE tree clusters separated samples from the northern Dinaric Mountains from those of the southern Dinaric Mountains into 2 clusters according to geographic location. The results obtained using a Bayesian clustering methodology was similar. By using mtDNA variation in chamois from Slovenia, Croatia, and Bosnia and Herzegovina, the existence of alpine chamois haplotypes in northern areas and Balkan chamois haplotypes in southern areas was confirmed. These results confirm the impact of recent human management (i.e., translocation) into the Velebit Mountains, which established a new contact (hybridization) zone between the subspecies. Therefore, future translocations must be planned carefully to avoid compromising genetic integrity and posing a serious risk to native species, as in this case.