Resumo. AIM: This study reports an investigation of limnological characteristics and aquatic macrophyte occurrence in a neotropical reservoir in order to assess the spatio-temporal variation of water and sediment variables and their influence on plant distribution. METHODS: Macrophytes, water and sediment samples were collected from a Brazilian reservoir in different seasons from four main arms of the reservoir. In total sixteen water-sediment variables were analyzed including N:P ratio and Trophic State Index. The plants were collected using a quadrat sampling procedure and the dry weight per sample was measured. MANOVA was performed to evaluate spatial and temporal variation of environmental variables as well as seasonal biomass differences. To assess the relationship among environmental variables and macrophytes an ordination analysis (using Canonical Correspondence Analysis: CCA) was carried out. RESULTS: The spatial and temporal variation of limnological variables generated a heterogeneous system which supports the presence of different species of macrophyte. pH, dissolved oxygen and sediment composition were important predictors of Polygonum lapathifolium occurrence while nutrients were associated with Eichhornia crassipes and Pistia stratiotes. Inorganic substances were related to biomass variation of Eichhornia azurea and Myriophyllum aquaticum. CONCLUSIONS: The spatial variation of the environmental variables has caused heterogeneity in the reservoir and it may support the occurrence of different species of macrophyte. Limnological variables highlighted in CCA are important to predict the species occurrence and their control in the study area.