Resumo. Abstract Aim: In this study, we analyzed the structure of the copepod community along a longitudinal axis of the last undammed stretch of the Upper Paraná River floodplain, downstream of Porto Primavera reservoir. We hypothesized that (i) copepod assemblage would show an increase in the abundance of organisms, species richness and specific diversity with the distance from the Porto Primavera reservoir, due to the arrival of species from adjacent lakes and tributaries; (ii) copepod abundance and species richness would be higher in tributaries and adjacent lakes located at the floodplain, which show higher environmental heterogeneity and function as a species source; and (iii) spatial patterns of copepod assemblage structure are related to the environmental gradient, since copepods show a rapid response to the variation of environmental conditions. Method Sampling was performed quarterly from August 2013 to July 2014, at the subsurface of the pelagic region. Sampling sites were located along the Paraná River, in seven of its tributaries, and in eight lakes located in its floodplain. Results We identified 29 species belonging to two families: Cyclopidae (21 species) and Diaptomidae (eight species). On contrary of our first hypothesis, ANOVA results showed a significant decrease in copepod abundance in the downstream direction. Similarly, the tributaries showed higher values of ecological attributes in the upstream stretch. RDA results showed that in axis 1, the Paraná River and the lakes were positively correlated with conductivity, pH, temperature and suspended organic matter, characterized by the most abundant copepod species. The tributaries, on the other hand, were negatively correlated with turbidity, total nitrogen, total phosphorus and depth. Axis 2 showed a positive correlation of the Paraná River with conductivity and suspended organic matter, and a higher abundance of Argyrodiaptomus azevedoi, Notodiaptomus iheringi, N. henseni, N. cearensis and N. cf. spinuliferus. On the other hand, the lakes were positively correlated with temperature and pH and also with a higher abundance of Thermocyclops minutus and T. decipiens. Conclusion We highlight the importance of the maintenance of the ecological integrity of the Upper Paraná River floodplain, especially of its tributaries, in order to support the high biodiversity registered in the last undammed stretch. Furthermore, we highlight the importance of manipulation of water levels in reservoirs located upstream of this stretch, a necessary condition to maintain the hydrological connectivity between floodplain habitats and the main river.