This paper purposed to evaluate alterations in fish assemblage (structure and species richness) and variations in the abundance of the most abundant species captured in the Piquiri River, due to the closure of Porto Primavera (in 1998; located upstream the Piquiri River mouth) and the Itaipu (in 1982; located downstream) dams in the upper Paraná River. The effects of selected environmental variables were also determined. Fishes were sampled during four distinct periods. Six samplings were conducted in each period, at two different sites (Campina and Apertado). For sampling, gill and trammel nets were used. Species abundances were indexed by catch per unit of effort. Spatial and temporal changes in environmental variables were summarized by a principal components analysis (PCA). To summarize structure of the assemblage, a nonmetric multidimensional scaling (NMS) was used. To test the significance of between‐group differences at each periods and sites we used a multiresponse permutation procedure (MRPP). To test the association between fish assemblage and the environmental variables matrices, we used procrustean superimposition approach. A total of 2693 individuals were caught, belonging to 69 species, 4 orders and 19 families. Species richness differed spatially; the Campina site had greater mean richness (13) per sampling than the Apertado site (10). The structure of the fish assemblages showed significant temporal and spatial variations, which were more evident in the last period at the Apertado site (after the closure of Porto Primavera Dam). Spatial and temporal changes in structure of fish assemblages were not associated to environmental variables. Temporal changes in abundance, both in number and in weight, of the most abundant species were verified in both sites. The lack of migratory species was highlighted, which sharply decreased in the last studied period, when they were replaced by sedentary species. These findings appear to be related to regional factors, such as the decrease of floods registered in the upper Paraná basin, exacerbated by the control prompted by dams. Copyright © 2009 John Wiley & Sons, Ltd.