The status of golden loaches (genus Sabanejewia) in the region of Central Europe and Balkans is still ambiguous. The greatest controversy is caused by species Sabanejewia balcanica and S. bulgarica, whose distinguishing features are often difficult to prove and their occurrence in some river sections is often mentioned as sympatric. Previous phylogenetic studies aimed at the resolving of their taxonomic status did not include samples from their type localities and so led to a lack of their true distribution in this region. Therefore, the main aim of this study was to identify the taxonomic status of golden loaches populations in this area on the model territory of Slovakia. For this purpose, we used a novelty approach (morphological, molecular and microhabitat) and we also included the missing samples from the type localities of both species. Based on mtDNA all the Slovakian samples reflected haplotype richness revealed on the type locality of S. bulgarica, although the genetic distances from other representatives of the genus Sabanejewia occurring are not significant. Within the morphology, we have revealed a great measure of variability in studied populations, which is largely caused by different habitat conditions and thus representing a phenotypic plasticity of these fish.