This study aimed to evaluate the fish fauna composition and distribution in different environments, up- and downstream the Gavião Peixoto Dam, in the Jacaré-Guaçu River basin. Most of the sampled ichthyofauna is autochthonous, and the majority of the species belongs to the orders Characiformes and Siluriformes. The order Characiformes comprised most of the specimens caught, Astyanax altiparanae being the most abundant species. All the studied environments were characterized by the massive occurrence of accidental species and low similarity between their ichthyofauna. In general, a greater number of species and higher values of the diversity index were observed downstream of the dam. The fish diversity in the studied habitats was positively correlated with the water body depth, and negatively correlated with the water conductivity. This last correlation may be an indicative of the negative influence of the pollution of the Jacaré-Guaçu basin on the local fish fauna.