Resumo. AIM: The objective of the present study was to demonstrate the influences of the environment spatial heterogeneity on benthic macroinvertebrates considering transverse and longitudinal gradients as also seasonality. METHODS: Four samplings were performed: two in the wet and two in the dry season in the riverine, transitional and lacustrine zones in the littoral and profundal regions of Itupararanga reservoir, SP, Brazil. Abiotic characterization of the water and of the sediment was performed. The biotic characterization was based on richness, dominance, diversity, and density of organisms, as well as on the relative abundance of predominant taxa. Two-way ANOSIM analyses were performed for both biotic and abiotic components, in order to test the significance of the differences in the longitudinal and transverse directions as well as of the differences between seasons. RESULTS: Compartmentalization was present in both directions, longitudinal and transverse. In a general way, the littoral region presented higher diversity values when compared with the profundal region, and the riverine zone presented high densities and high percentage of taxons, which usually indicate organic pollution. The differentiation between the transitional and lacustrine zones was determined mainly by taxonomic composition. Seasonality was also observed and the transportation of small particles, the entrance of nutrients, and the presence of macrophytes were considered as determinants for differentiation. CONCLUSIONS: Together, these results demonstrate the responses of benthic macroinvertebrate communities considering distinct sources of variation: longitudinal heterogeneity, determined by the increasing distance from the forming rivers that leads to a gradient of physical and chemical conditions; transverse heterogeneity, determined by the proximity with the land environment and depth differences. Seasonal heterogeneity was recorded during the period of this research and it was determined by variations in water flow, in temperature, and in the carrying of matter, nutrients and organisms.