A karyotypic analysis was carried out using conventional staining and C-banding in 32 specimens of Astyanax scabripinnis (Pisces, Characidae) from Campos do Jordao (Sao Paulo State, Brazil). Twenty-eight individuals (87.5 per cent of the sample studied) showed 1–2 extra B-chromosomes, similar in size and shape (metacentrics) to pair 1 of the karyotype, with a high intra-individual constancy. Two types of B could be identified on the basis of their constitutive heterochromatin patterns. The hypothesis of the origin of the B-chromosome from non-disjunction of chromosome 1, followed by a heterochromatinization process, is postulated.