||Delineating biogeographical regions is a critical step towards the establishment and evaluation of conservation priorities. In the present study, we analysed the distribution patterns of the freshwater fish of an understudied European biodiversity hotspot, the Balkan Peninsula. Based on the most extensive available database of native freshwater fish species distributions, we performed a hierarchical clustering analysis to identify the major biogeographical regions of the Balkan Peninsula. We also highlighted the 'hottest hotspots' of freshwater fish diversity across the delimited biogeographical regions by describing the patterns of species richness, endemic and vulnerable species; indicator species were also determined. The bioregionalisation scheme consisted of eight groups of drainage basins that correspond to distinct regions of the Balkan Peninsula. Overall, the delineated biogeographical regions varied in terms of species richness, endemism, vulnerability (i.e. extinction threats) and indicator species composition. From a conservation perspective, this study emphasises the prioritisation of areas characterised by high levels of irreplaceability (endemism) and vulnerability (i.e. the Attikobeotia region, Ionian Sea and Prespa Lakes) and stresses the necessity of implementing a network of protected freshwater areas across the Balkan Peninsula.