Abstract We analyzed the karyotypes of three specimens of fishes of the genus Hypostomus Lacepede, 1803 (Loricaridae) from Xingu River (Amazon region). We used conventional staining techniques, including C-banding, Ag-NOR staining, CMA3- and DAPI-banding. Each specimen had a different cytotype: Hypostomus sp. Xingu-1 had 2n=64 (32M/SM, 32ST/A); Hypostomus sp. Xingu-2 has 2n=66 (32M/SM, 34ST/A), and Hypostomus sp. Xingu-3 had 2n=65 (38M/SM, 26ST/A + 1 B). The three cytotypes showed similar C-, CMA3- and DAPI-banding patterns. The nucleolus organizing regions were located in the short arm of chromosome pair 25 of Hypostomus sp. Xingu-1 and pair 29 of Hypostomus sp. Xingu-2, and in the long arm of pair 30 of Hypostomus sp. Xingu-3, probably because of a pericentric inversion. A fusion/fission rearrangement explains the difference in the diploid number and number of M/SM and ST/A chromosomes between the 2n= 64 and 2n=66 cytotypes. The B chromosome most probably explains the difference between the 2n=64 and 2n=65 cytotypes. The cytotype with 2n=65 had a significantly larger number of M/SM chromosomes, probably because of pericentric inversions. These three cytotypes may represent different species.