Abstract – We examined the relative contribution of environmental heterogeneity and dispersal limitation on freshwater fish community composition in 18 Greek protected lakes and reservoirs. Environmental heterogeneity was measured by spatial pattern metrics (calculated by digital map processing, e.g., patch richness density, area-weighted mean patch area), altitude, maximum lake depth and trophic status. Dispersal limitation was measured by straight-line distances among lake centres. Ecosystems were clustered according to species composition. We examined the correlation of similarity in species composition among ecosystems with that of environmental heterogeneity and with straight-line distances, for the entire dataset, as well as for the occurring clusters. Fish species richness increased with ecosystem area and decreased with altitude. The clusters identified (aquatic ecosystems of Northern vs. ecosystems of Western Greece), implied an underlying biogeographical pattern as defined, with Pindus range acting as a natural barrier. Between ecosystems similarity, based on fish species composition, showed a weak to insignificant correlation with environmental heterogeneity, but was significantly correlated to dispersal limitation for the entire dataset as well as within each occurring cluster. Thus, natural barriers, species biogeography and dispersal limitation played a more significant role in shaping freshwater fish communities than environmental heterogeneity.