In Bulgaria, chamois (Rupicapra rupicapra balcanica) are protected by law and it has been recommended to assess their genetic diversity, level of inbreeding, and extent of introgression by Alpine chamois (R. r. rupicapra) that were released in the last century in the Rhodope Mountains. Chamois (n = 81) collected between 2009 and 2013 at the "Izvora State Hunting Reserve" (ISHR, western Rhodopes, a major source for chamois translocations in Bulgaria; census size: 200-250) were screened for allelic variability at 12 microsatellite loci and compared to chamois (R. r. rupicapra, n = 135) from six populations in the Austrian Alps. Indices of genetic variability were significantly lower for the ISHR population than for the Austrian populations. A significant bottleneck signal was observed for the ISHR population, but no distinct inbreeding signal due to non-random mating. F-statistics, absolute genetic distances, Bayesian STRUCTURE and assignment analyses, and a factorial correspondence analysis concordantly indicated distinct differentiation between the ISHR and Austrian chamois. STRUCTURE identified some few ISHR chamois as possibly introgressed by Alpine chamois. ONeSAMP revealed effective population size estimates of Ne a parts per thousand 50 for the ISHR population. A reduction of shooting quotas, a reinforcement of poaching control, and exchanging chamois with other Bulgarian populations should increase its Ne to prevent further loss of genetic variability. Screening for introgression should be extended to other Bulgarian populations, particularly to areas where Alpine chamois were released in the past; and it should accompany all translocations, to prevent possible dissemination of introgressed chamois.