Cytogenetics studies on Astyanax sp B, an endemic species of the Iguaçu River (Paraná, Brazil) were carried out. The specimens showed a standard diploid number of 2n=50 chromosomes with a karyotype consisting of 6 metacentric, 24 submetacentric, 6 subtelocentric and 14 acrocentric chromosomes. In addition to the basic karyotype, all the specimens presented 1–2 supranumerary or B chromosomes in mitotic metaphases. Three morphological types of B chromosomes have been identified in population under analysis: an acrocentric-type microchromosome; a metacentric macrochromosome smaller than the first pair of the normal karyotype complement; a small submetacentric chromosome. C-band analysis shows discrete markings in the centromeric regions of various chromosomes which contrast with strongly heterochromatic blocks in the telomeric regions of the long arms of chromosome pairs 20, 21 and 22 (acrocentric). Heterochromatic block-sized heteromorphism has been among homologues of pair 20. The chromosome characterisitics of Astyanax sp B indicate similarity with other species of the genus Astyanax with regard to karyotypic macrostructure, distribution of constitutive heterochromatin (C-band pattern) and occurrence of B chromosomes. Some aspects related B chromosomes in this species are discussed.