Global warming, eutrophication, fisheries overexploitation, species invasions, and habitat loss are the major threats to freshwater biodiversity. The aim of this study was to determine the species richness and diversity of fish in 535 Polish lakes of different morphometry (area of more than 50 ha and a maximum depth ranging from 0.4 to 108.5 m) and trophic status (from oligotrophy to hypereutrophy). A total of 39 fish species were found in the studied lakes, among which eight species were alien invasive. The Shannon diversity index varied between 0 and 2.04.The most common and frequent species were Rutilus rutilus (99.8% frequency) and Perca fluviatilis (99.6% frequency). Ten fish species, including five alien ones, were characterized by a very low frequency (<1%). The number of fish species in single lakes ranged from 1 to 19. In most of the studied lakes, 11 and 12 species (104 and 108 lakes, respectively) were caught. The richest taxonomic composition (19 species) was recorded in the meso-eutrophic lake with an area of 80 ha and a maximum depth of 34.4 m, slightly poorer (18 species) in the hypereutrophic lake with an area of 168 ha and a maximum depth of 2.7 m. The poorest taxonomic composition (one species) was found in a lake with low conductivity and circumneutral pH. Statistical analysis showed that the number of fish species decreased with increasing eutrophication conditions of the studied lakes, while it increased with an increasing area and the maximum depth of these lakes.