Cytogenetic analyses were performed on three Gymnotus species, Gymnotus paraguensis, Gymnotus sylvius and Gymnotus pantanal, collected in sympatry in the region of Guaíra, Paraná State (Brazil). Gymnotus paraguensis presented 54 chromosomes (52m-sm + 2st) in both sexes, G. sylvius presented 40 chromosomes (36m-sm + 4a) in both sexes and G. pantanal presented 40 chromosomes (14m-sm + 26a) in females and 39 (15m-sm + 24a) in males, with an X1X1X2X2/X1X2Y multiple sex chromosome system. All three species had single nucleolar organizing regions and heterochromatin in the centromeric region of most chromosomes. Gymnotus pantanal, previously collected only in the Lower Paraná and Paraguay River basins, was confirmed for the first time in the Upper Paraná River basin. This species constitutes one of several examples of species that have colonized the region after flooding of the great Sete Quedas waterfalls by the construction of the Itaipu hydroelectric dam. The probable absence or infertility of hybrids is indicated by the high level of chromosome differentiation between these species.