AIM: The present study analyzed the spatial variation in fish species richness and composition in streams of the Ivinhema River basin, Upper Paraná basin, determining the relationship between local and regional descriptors of the fish assemblage organization; METHOD: From 2001 to 2011 we sampled 200 stream stretches, using different sampling methods. Local species richness and composition were analyzed using univariate and multivariate methods to detect patterns of variation in the fish assemblage organization; RESULTS: We identified 111 fish species in the streams, and estimated the occurrence of 117 fish species (CIα0.05 = 111 to 123) with lower species richness in headwater streams compared to the lower parts of the basin. The fish species recorded were predominantly of small size, and the mean size increased from the headwaters to the river mouth. The four most common species are widely distributed in the basin. However, 24 species are allocthonous to the Upper Paraná basin, and two species (Chaetobranchopsis australis and Apistogramma commbrae) are recorded here for the first time in the Upper Paraná basin. Altitude was the main determinant of local fish species richness and composition, and a discontinuity in assemblage organization (richness, composition, fish size) was detected at approximately 430 m a.s.l.; CONCLUSION: Streams of the Ivinhema River basin are inhabited mainly by small-sized fishes; and longitudinal variation, interacting with local characteristics, determines the fish assemblage patterns.