This study investigates the distribution and structure of the assemblages of fishes in tributaries of middle and lower sections of Tibagi river basin. The samples were conducted quarterly from December 2013 through December 2014, in 24 sampling sites of 8 tributaries. A total of 5643 individuals belonging to 44 species distributed in 13 families and 5 orders are reported. The families Characidae, Loricariidae, and Heptapteridae were predominant; Astyanax paranae, Phalloceros harpagos, Piabina argentea, Astyanax bockmanni, and Trichomycterus davisi were the most abundant species. Four non-native species were collected: Coptodon rendalli, Poecilia reticulata, Xiphophorus hellerii, and Oreochromis niloticus. The fish fauna of the middle and lower sections of Tibagi river basin has previously been threatened by human activities such as urbanization, industry, agriculture, and currently with dam construction.