Chromosomal characterization of constitutive heterochromatin was performed in Astyanax sp. D, a species found in the Iguaçu river, using C-banding, fluorescent in situ hybridization (FISH) of the repetitive DNA family As51, localization of the 18S rDNA sites and base-specific fluorochromes. The sites of the satellite DNA coincide with large heterochromatic blocks present in this species, namely CMA3- and DAPI-. The many 18S rDNA sites are CMA3+. Similarity between the large heterochromatic blocks is suggested, as well as the identification of two types of heterochromatin, one related to the 18S rDNA domains and the other to the As51 regions.