The upper rio Paraná basin drains the most developed and environmentally degraded region in South America: the Brazilian southeast. While it is one of the most well-known Neotropical fish assemblages, it is also one of the most threatened by anthropic activity. Urbanization, deforestation, dam construction, invasive species, and water pollution not only reduce the living area of species but also alter our perception of the basin and its biotic elements. Such changes are so profound and pervasive that raise uncertainties about the native or non-native status of several species. This paper aims to offer a detailed picture of the native diversity and historical biogeography of the fishes in the upper rio Paraná basin, which we hope will offer a solid foundation for future conservation policies. We update the list of native species, analyze geographical distributions, and identify biogeographical patterns, emphasizing areas requiring recognition as distinct biotas for conservation efforts. Over the past 15 years, more than 100 additional species have been described or recorded, expanding the total to 341 native species belonging to six orders and 30 families, making it the richest river basin in Brazil outside the Amazonian region. Unlike most neotropical basins where Characidae dominates, loricariids make up nearly one-fourth of the total fish diversity in the region. Species and clades concentrate in the central basin, while endemics are confined to peripheral areas due to the complex biogeographical history shared with neighboring basins. Eighteen distinct biogeographical regions are identified, discussing their composition, histories, and conservation implications. The upper rio Paraná basin has 10% of its fish fauna ranked as endangered and present 128 non-native species, including three hybrids. This places it as the major drainage with the largest number of endangered taxa in the neotropics and at the same time the one with the most numerous non-native elements. Existing protected areas are deemed ineffective in preserving diverse assemblages and fail to safeguard the majority of threatened and narrowly-endemic species.
