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). Freshwater fish assemblages are structured by variables related to both water quality and riparian vegetation 1-6. In this sense, warmer waters exhibit higher fish abundance and biomass while highly oxygenated waters may lead to greater species diversity 7-9. Riparian vegetation is a transitional semiterrestrial system 10 that provides energy in watercourses through the input of organic matter 2. Leaves deposited on the watercourse bed contribute indirectly to fish food because they act as a substrate for numerous microorganisms 11 and insects 12,13. In addition, riparian trees and roots restrict channel widening, cause channel deepening and add coarse woody debris favoring fish concealment and channel complexity. The influences of water and riparian vegetation on fish assemblages are not independent 2,10,14,15 ; that is, riparian vegetation may directly or indirectly influence water variables 16. For example, water temperature is directly influenced by riparian vegetation, which regulates the watercourse insolation level 17,18 and influences primary production 19. Conversely, channel depth and substrate heterogeneity are indirectly influenced by riparian vegetation because the riparian zone regulates the entry of sediment that can be deposited into the watercourse 10,20,21. Another factor that should not be neglected is the spatial factor (e.g., the river network), which includes geographical barriers that hamper or prevent species migration between locations. Abundance and richness are diversity metrics that are spatially structured 22-28. Spatial factors are a consequence of the geological and local climatic influence on the streams in a river network 29-31 and the position of the watercourse along a longitudinal gradient (upstream-downstream 32) for the 1 st...