Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) ; Conventional (Giemsa, C-banding, Ag - NORs) and molecular [5S rDNA, 18S rDNA, (TTAGGG)(n)] cytogenetic techniques were employed to study six species of the genus Eigenmannia Jordan & Evermann, 1896. They exhibited diploid chromosome numbers ranging from 2n=28 (Eigenmannia sp. 1) to 2n=38 (E. virescens (Valenciennes, 1836)). The C-banding results revealed that species with the lowest 2n have less heterochromatin content and that morphologically differentiated sex chromosomes observed in two species showed distinct patterns of heterochromatin. While the X-1, X-2 and Y-chromosomes of Eigenmannia sp. 2 showed only centromeric heterochromatin, the XY sex chromosomes of E. virescens possessed large heterochromatic blocks in the terminal position, particularly on the X chromosome. The nucleolus organizer regions (NORs) were located in different positions when compared to the 5S rDNA sites. Additionally, the presence of minor ribosomal gene sites on the sex chromosome pair of E. virescens represented a new type of the sex chromosomes in this group. The telomeric probe (TTAGGG)(n) hybridized to the terminal portion of all chromosomes in all species examined however, interstitial telomeric sites were found in the metacentric pair No. 2 in Eigenmannia sp. 1. The analyzes confirmed some hypotheses about karyotype evolution in the genus Eigenmannia, and brought new information about the distribution of the genetic material in the chromosomes of the samples analyzed providing new insights for understanding the process differentiation in the genomes of species under study.