ABStRACt. Hypostomus sp 3-Córrego Salobrinha NUP 4247 and Hypostomus sp 2-Rio Perdido NUP 4249, collected in the Planalto da Bodoquena, Paraguay River basin, Brazil, were characterized cytogenetically. Hypostomus sp 3-Córrego Salobrinha showed two modal numbers. This polymorphism consists of the presence of two extrachromosomes. It was not possible to define the diploid number in four specimens, where cell lineages had 2n = 83 and 2n = 84 chromosomes in one individual, and 2n = 82, 2n = 83 and 2n = 84 chromosomes in the others. These results reveal the existence of a genetic mosaic due to the occurrence of one or two extrachromosomes in this species. Hypostomus sp 2-Rio Perdido NUP 4249 showed a 2n = 84, FN = 106 with size heteromorphism in one pair of chromosomes stained with AgNO 3 . In both species, C banding showed a pattern of heterochromatin distribution with a few small bands in the centromeric and pericentromeric regions coinciding with chromomycin A 3 staining. Until now, the major diploid number for the genus Hypostomus was 2n = 80, but the species studied here had chromosomes that in- creased this number and the variation for this genus. Our results are also the first cytogenetic data on Hypostomus from the Paraguay River basin.