The genus Astyanax is a specious Neotropical fish group, occurring from the south area of the United States to Argentina. During the past few years, intensive studies on representatives of this group have been performed, broadening the genetic, taxonomic and biogeographical knowledge considerably. However, phylogenetic relationships among many species remain uncertain. Significant karyotypic variations occur in A. bimaculatus, A. scabripinnis and A. fasciatus, suggesting the existence of species complexes. Samples with different diploid numbers may be found in A. fasciatus and, in some cases, morphological differentiation among them is not easily verified due to the absence of clear diagnostic characteristics. In the present study, the organization of the A. fasciatus genome from the Tietê River, Mogi das Cruzes, SP, Brazil, was analyzed, describing the karyotype structure and the chromosomal mapping of 18S and 5S ribosomal DNA (rDNA), H1 and H3 histones genes, telomeric DNA, and Rex3 transposable element. Sequencing of the cytochrome c oxidase I (COI) gene was also performed. Chromosomal analyses highlighted the occurrence of 2 distinct karyomorphs in the referred population, one with 2n = 46 and the other with 2n = 48 chromosomes. Furthermore, physical mapping of the repetitive sequences and nucleotide analysis of the COI gene reinforce the genomic differentiation between those karyomorphs and corroborate the existence of 2 different lineages morphologically similar to A. fasciatus living in sympatry.