Riparian vegetation plays an important role in providing energy to small watercourses and maintaining ecological processes through organic matter input and together with hydrological and geomorphological watercourse characteristics influence on fish assemblages. The goal of this paper was partitioning and quantifying the influence of riparian zone (type of riverbank substrate, bank slope, type of riparian vegetation cover and percentage of riparian vegetation cover on the main channel), physical habitat (stream channel width and depth, type of substrate and aquatic habitat in channel, water velocity and organic matter), water quality (turbidity, temperature, conductivity, pH, dissolved oxygen and chlorophyll concentration) and spatial variables (linear distances between sampled points) on fish assemblages (richness and abundance per species) in headwater streams of the Upper Paraná River basin, Central Brazil. For this purpose, it was performed a variation partitioning analysis between riparian, physical habitat, water and spatial variables sets and a Redundancy Analysis to quantify the influence of variables on the fish assemblages. Only the physical habitat and water quality variables influenced the fish assemblages (richness and abundance per species).