Four populations of Astyanax scabripinnis (Pisces, Characidae) were analyzed for B chromosome features. This species contains a metacentric macrochromosome (BM), similar in size to the first chromosome of the karyotype, besides two variant forms, a large submetacentric (BSM) and a small metacentric (Bm), both showing a reduced frequency. These variant forms were observed only in the populations from the Campos do Jordão region (São Paulo State, Brazil), although not present in all the populations sampled. The three B chromosome forms are heterochromatic and at least the BM and BSM are also GC-rich, as evidenced by C-banding and chromomycin A3 staining. In spite of the morphological similarity between the BM form and the first chromosome pair, they differ in the G-banding pattern, which does not favor a possible relationship among these chromosomes. FISH with a telomeric DNA probe evidenced signals on the terminal region of all chromosomes, including the Bs. Interstitial telomeric bands, indicative of some chromosomal rearrangements such as translocation or tandem fusion in the origin of the B chromosomes, were not observed. BSM and Bm are probable derivative B chromosome forms from an ancestral BM chromosome, showing a restricted occurrence and low frequency in the populations.