Rivers on the Balkan Peninsula can be separated into ichthyofaunistic areas with different endemic fish species. The Vardar River contains a particularly large number of endemics, indicating its complete and long-term isolation from neighbouring river systems. One of the few species shared with other rivers is the loach species Oxynoemacheilus bureschi. In this study, the genetic analyses of 175 individuals of O. bureschi from 17 sites, covering the entire distribution of the species, including the Rivers Vardar (= Axios), Struma (= Strymon), Mesta (= Nestos) and Danube, were performed using one mitochondrial and one nuclear marker. Genetic differentiation among populations was in general low. Shared haplotypes were common and occurred even between distant localities and different river systems. This points to a high degree of gene flow among populations and rejects the hypothesis that the population in the Vardar River represents a relict from an early colonization of the Balkan Peninsula. In contrast, the results suggest that populations in the Vardar River, as well as those in the Danube River, are of recent origin, and a human-mediated introduction cannot be excluded. On the other hand, the populations in the Aggitis River, a left tributary of the lower Struma River, were clearly separated from the rest of the species and represent a long-term isolated lineage. Demographic analyses suggest a recent population expansion for O. bureschi, in which the population in the Aggitis River was not involved.