The Persis and Hormuz basins in southern Iran, due to hydrological and biogeographical conditions, are among the most important habitats for freshwater fish in the country. This study aimed to investigate beta diversity patterns of freshwater fish and identify biodiversity hotspots in these two basins. Sampling was carried out using electrofishing equipment in various rivers of the two basins during 2025, and species occurrence data, after removal of duplicate records, were analyzed in the form of a presence–absence matrix. To calculate beta diversity, the Jaccard index was used, decomposed into species replacement (turnover) and richness difference components using the adespatial package in R software. The results showed that fish communities in the two basins exhibit high biotic heterogeneity, with both the species replacement and richness difference components playing important roles in shaping diversity patterns. The identified specimens belonged to 16 families, 34 genera, and 48 species. The species Capoeta saadii, present at 73 stations, was the most widespread species in the region, whereas 10 species were recorded at only a single station, indicating their restricted distributions and high vulnerability. Beta diversity attributable to species richness differences, as well as total beta diversity, exhibited the highest values in the Persis Basin and the lowest values in the Hormuz Basin, whereas the contribution of the species turnover component to total beta diversity was negligible. The results of this study indicate that hydrological differences, geographic isolation, and specific environmental conditions play a determining role in shaping the fish community structure of these basins, and that protecting the sensitive habitats of these regions is essential for preserving the freshwater fish biodiversity of Iran.