The chamois is a useful species with which to investigate the combined genetic impact of habitat fragmentation, over hunting, and translocations. Genetic variation within and between chamois (genus Rupicapra) populations was analyzed in 259 individuals from 16 sampling sites located in Italy, Spain, Slovakia, and the Czech Republic. Two mitochondrial DNA markers (control region and cytochrome b) and 11 nuclear microsatellites were typed. The principal results of this study can be summarized as follows: 1) high and significant differentiation between almost all chamois populations is observed even on a microgeographical scale, probably caused by the patchy distribution of this species, sharp geographical barriers to gene flow, and drift effects related to recent bottlenecks; 2) historical translocation events have left a clear genetic signature, including interspecific hybridization in some Alpine localities; 3) the Apennine subspecies of chamois, Rupicapra pyrenaica ornata, shows a high and similar level of divergence (about 1.5 My) from the Pyrenean (Rupicapra pyrenaica pyrenaica) and the Alpine (Rupicapra rupicapra) chamois; therefore, the specific status of these taxa should be revised. These results confirm the potential of population genetic analyses to dissect and interpret complex patterns of diversity in order to define factors important to conservation and management.