Resumo. Abstract Aim: This study aimed to characterize the zooplankton structure in highland streams in southern Brazil, Pelotas River sub-basin, as well as the respective relationships with the environmental conditions in the different seasons. Methods Zooplankton was collected using a plankton net (68 µm mesh) and concentrated into a volume of 50 ml for later analysis in the laboratory. Sampling occurred during the winter (August 2013), spring (November 2013), summer (February 2014) and autumn (May 2014). Results Rotifers and testate amoebae were the predominant groups. Mean abundance, diversity and evenness varied considerably between the different seasons. The variability of zooplankton was correlated to environmental conditions (conductivity, suspended solids, pH, temperature and dissolved oxygen). Conclusions The seasonal variation of the zooplankton community made it possible to elucidate some questions involving the ecology of these organisms in highland streams in southern Brazil. These unique ecosystems warrant further research and monitoring efforts, including studies of their hydrological patterns, biological diversity and adaptive mechanisms of the zooplankton community.