<p>The southwestern Balkans have an exceptional level of freshwater fish endemism, with the Ionian freshwater ecoregion recognized as a key biodiversity hotspot there. In this study, we examined how river Fish Assemblage Types (FATs), identified from 175 electrofished river sampling sites, shape the spatial patterns of β-diversity in this ecoregion. Initially, we used bipartite networks to identify areas of distinct fish assemblages. Subsequently, we used the Ružička index to estimate total β-diversity, decomposing it into the components of replacement and abundance differences. Local (LCBD) and species (SCBD) contributions to β-diversity were calculated. By applying generalized dissimilarity models and generalized additive models, we reveal which regional or local variables shape β-diversity and influence species important to the β–diversity patterns. Six distinct FATs were identified, each exhibiting high levels of β-diversity, but differing in the β-diversity component that contributed the most to the overall dissimilarity pattern. Geographic distance and temperature had the greatest influence on β-diversity, reflecting long-term geographical isolation or past connectivity among river basins, and a longitudinal river zonation pattern. The results provide insights into fish community and biophysical nuances that should be considered when building conservation strategies for this ecoregion. In the headwaters where rheophilic cold water species dominate, conservation actions should focus on protecting specific habitats and cool water conditions. In the middle and lowland areas, mixed methods should address both spatial replacement (endemic species) and abundance differences (widespread species). The findings of this study contribute to the identification of ecologically important water bodies and support conservation efforts by enhancing our understanding of spatial diversity patterns.</p>