Background: The damselfishes, an extremely diverse group of herbivorous fish, stands out as an important and ubiquitous ecological component of coral reefs. In the Western South Atlantic, the genus Stegastes is the most representative, whose evolutionary paths and taxonomic status of insular endemic species have been better evaluated. To clarify the karyotypic evolution involved in the diversification of this group, cytogenetic analyses were performed in four nominal species (Stegastes variabilis and Stegastes fuscus, distributed in Brazilian coastal regions; Stegastes rocasensis and Stegastes sanctipauli, from Rocas Atoll and São Paulo and São Pedro Archipelago) and one subspecies (S. fuscus trindadensis, from Trindade and Martim Vaz Archipelago). Results: Classical cytogenetic protocols and fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes were used for comparative analyses. All species had 2n = 48 chromosomes, with high FN values ranging from 88 to 92. Stegastes rocasensis and S. sanctipauli shared identical cytogenetic patterns, while S. f. trindadensis revealed a syntenic arrangement of 18S and 5S rDNA sites not found in S. fuscus from the Brazilian coast. Conclusion: The karyotypic evolution of Stegastes was predominantly driven by multiple pericentric inversions (and/or centromere shifts), resulting in changes in the internal organization of chromosomes. S. rocasensis and S. sanctipauli have similar cytogenetical patterns, as well as S. fuscus and S. f. trindadensis indicating incipient evolutionary differentiation in insular species. Mapping other repetitive DNA sequences provided an exceptional opportunity to clarify chromosomal changes and their association with the evolutionary diversification of Stegastes species.