Cytogenetic data obtained for Serrasalmus rhombeus revealed two cytotypes (cytotype 1 and cytotype 2) at four sampling sites. Cytotype 1 consists of 2n = 60, 20M 24SM 6ST 10A and was detected in specimens from Negro River (Anavilhanas Islands), Solimões River (Camaleão Lake), confluence of Negro and Solimões Rivers (Catalão Lake), and Uatumã River. Cytotype 2 consists of 2n = 58, 22M 24SM 2ST 10A and was detected in specimens from Catalão and Camaleão Lakes. In Cytotype 2, the first chromosome pair was almost twice the size of second pair, and the constitutive heterochromatin pattern of each cytotype is distinct. The two cytotypes occurred sympatrically at Catalão and Camaleão Lakes, and no intermediates between the cytotypes were detected, suggesting each cytotype represents a different fish species.