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Abstract Aim: We investigated the spatial and temporal patterns of abundance and diversity of planktonic ciliate community, in the last undammed stretch of the Upper Paraná River, Brazil. Methods In order to reach this result, seven field campaigns were performed over two years. Plankton samples were collected from 10 transects through this stretch of the river (230 Km), near the banks and on the center, as well on seven of its tributaries. Results 118 ciliate species were identified, among which the peritrichs were the most abundant while the order Hymenostomatida was the most specious group. We recorded a remarkable increase in abundance and species richness along the river, especially in the rainy period. Moreover, in this period we found an increase in the beta-diversity along the river, which consists in a remarkable distinction among the low, middle and high stretch of the river. In this way, continuous changes in the community structure of planktonic ciliates were evidenced, highlighting the importance of the precipitation and tributaries in the maintenance of the highest regional diversity in the studied area. Conclusion Our results strongly suggest the requirement for conservation actions with the purpose to maintain those tributaries undammed, in order to avoid biotic homogenization processes and the consequent reduction of aquatic biodiversity in this important neotropical ecosystem.