Abstract Biodiversity conservation often is aimed at areas of high species richness and endemism. Our understanding of freshwater fish diversity in the Neotropics is biased by lack of exploration within mountainous regions that historically have been difficult to access. These regions are particularly likely to contain high species endemism. The upper Mazaruni River drainage is hydrogeographically and climatically distinct from the rest of the Essequibo Basin. Recent ichthyological expeditions to the upper Mazaruni discovered up to 32 species previously unknown to science and a fish assemblage with perhaps the highest level of endemism in the Neotropics. This unique ichthyofauna is threatened by gold mining that has severely degraded aquatic habitats and therefore requires immediate protection.