Ecoregion delineations have gained increased attention in Europe, especially following the Water Framework Directive 2000/60/EC (WFD), which provides the European Union's first policy-relevant ecoregion map. However, the WFD's ecoregions were created through a minor adaptation of Illies' (Limnofauna Europaea. Gustav Fisher Verlag, Stuttgart, 1967/1978) freshwater zoogeographic regions, and the map's specific boundaries have not been widely evaluated with respect to the WFD's uses or their biogeographic accuracy. We examined the WFD ecoregion boundaries in Greece and its neighboring Balkan states by comparing them with the most prominent freshwater biogeographic boundaries as shown by riverine freshwater fish assemblages. Classification and ordination analyses of 23 river basin fish assemblages helped delineate natural faunal break boundaries in freshwater species assemblage distributions depicting major biogeographic barriers to aquatic biota dispersal. However, these biogeographic boundaries differ from those delineated in the WFD map, suggesting boundary errors and inconsistencies in the delineation method of the WFD ecoregions. We reviewed specific boundary disagreements and produced a map showing the region's most prominent freshwater biogeographic boundaries by charting them on watershed borders among the four biotically dissimilar river basin groups in the southern Balkans. This regional evaluation reveals both a need to reconcile disparate approaches to ecoregion mapping and to promote the development of a new policy-relevant inland waters ecoregion framework that would support broad-scale water management and aquatic conservation.