In the tropical Atlantic, the ichthyofauna between the coast of Brazil and the Caribbean regions, divided by the Amazon barrier, is very similar presenting several geminate species, including Gramma brasiliensis, endemic in Brazil, and its Caribbean counterpart Gramma loreto. Morphological and molecular studies have helped establish evolutionary patterns that sister-species of these two marine habitats are subjected to. However, their chromosomal characteristics are only beginning to be better characterized. Accordingly, a comparative cytogenetic analysis was carried out in G. brasiliensis and G. loreto, seeking evidence of cytotaxonomic markers implicated in the karyotypic diversiWcation of these species and likely associated with speciation events. Heterochromatic regions and their aYnity to Xuorochromes GC-or AT-speciWc were identiWed, as well as the distribution of ribosomal DNA sites in chromosomes, either by silver nitrate impregnation (Ag-NORs) or dual-color FISH mapping with 18S and 5S rDNA probes. While displaying the same diploid number, 2n = 48 chromosomes, considered basal for Perciformes, the two species diVered in karyotype structure, showing karyotypic formulas and species-speciWc heterochromatin pattern. The cytological characters found support the diVerentiating status of these species, possibly achieved under the conditions of allopatry due to the Amazon/Orinoco barrier, showing chromosomal peculiarities in Grammatidae species when compared to other groups of Perciformes.