The translocation of wild animal species became a common practice worldwide to re-establish local populations threatened with extinction. Archaeological data confirm that chamois once lived in the Biokovo Mountain but, prior to their reintroduction in the 1960s, there was no written evidence of their recent existence in the area. The population was reintroduced in the period 1964–1969 when 48 individuals of Balkan chamois from the neighbouring mountains in Bosnia and Herzegovina were released. The main objective of this study was to determine the accuracy of the existing historical data on the origin of the Balkan chamois population from the Biokovo Mountain and to assess the genetic diversity and population structure of the source and translocated populations 56 years after the first reintroduction. We used 16 microsatellite loci to analyse the genetic structure of three source chamois populations from Prenj, Čvrsnica and Čabulja Mountains and from Mt. Biokovo. Both STRUCTURE and GENELAND analyses showed a clear separation of the reintroduced population on Biokovo from Prenj’s chamois and considerable genetic similarity between the Biokovo population and the Čvrsnica–Čabulja population. This suggests that the current genetic composition of the Biokovo populations does not derive exclusively from Prenj, as suggested by the available literature and personal interviews, but also from Čvrsnica and Čabulja. GENELAND analysis recognized the Balkan chamois from Prenj as a separate cluster, distinct from the populations of Čvrsnica and Čabulja. This suggests that the Neretva River and the state M17 road are geographic barriers for the species dispersal, as they form a genetic boundary.