The fish fauna of Rio de Janeiro has been extensively studied, resulting in a comprehensive database of species collected over more than three centuries. This study aimed to identify fish species, their locations, and compile scattered information to aid in climate action and freshwater conservation prioritization and an evaluation of the sampling effort to date, as well as to identify patterns of diversity and distribution of freshwater ichthyofauna, delineate biogeographic units, and explore similarity relationships between areas. Analyzing data from nearly 25 ichthyological collections and literature on original species descriptions revealed 346 fish species: 172 freshwaters native, 22 allochthonous, and 152 marine species. The checklist includes updated species names. The sampling effort in Rio de Janeiro is high, especially in coastal lowlands. The findings indicate that inventory work is still needed in certain areas. Five bioregions of freshwater ichthyofauna were identified, along with six major areas of higher species richness. This biogeographic assessment underscores the diverse and distinctive freshwater fish fauna in the basins of Rio de Janeiro, with well-defined biogeographic units.