The genetic differentiation of Oxynoemacheilus bureschi (Drensky, 1928) from all three drainages (Struma=Strymon, Mesta=Nestos, Vardar=Axios) where this species occurs, as well as its phylogenetic relationships with other European stone loaches, was assessed using the mitochondrial cytochrome b gene. The phylogenetic reconstruction revealed two distinct clades within the European stone loaches with high bootstrap values. Very low genetic variability with no internal haplotypic structure has been found between and within all examined O. bureschi populations, indicated by low polymorphism and similar haplotypes. According to the nesting design and demographic patterns, the range of O. bureschi was not constant, but underwent expansion in the recent past. Lack of variation, a rather unusual phenomenon for fishes from the southern Balkans, is ascribed to facilitation of dispersal due to seashore regression, confluence events and river capture during the Pleistocene.