Fishes were collected at 36 sites in first order streams of Tibagi River basin, Paraná state, Brazil. A total of 2,669 individuals belonging to 47 species of 13 families and six orders were collected. The number of collected species represented 95.9% of the expected number of species for the studied area as estimated by Chao 1, and 93.4% by ACE, indicating an effective sampling. The highest richness was recorded in the tributaries at the lower region (Zone III) of the Tibagi River basin, with 33 species, including 16 species not found in the other regions of the basin. In the streams of the middle and upper regions (Zones II and I) each presented less richness, with 10 (two exclusive) and 25 (11 exclusive) species, respectively. This study shows a high geographic variation in the composition of fish fauna among zones, maybe related to historical and/or ecological influences.