Damselfishes (Pomacentridae, Perciformes) occur in all major oceans of the world and, with approximately 320 species, represent one of the most diverse families of marine Teleostei. The taxonomy of these reef fishes is problematic because of the large number of complex species and the range of color patterns they display, which vary among individuals and populations of the same species. In this study, we examined the cytogenetic composition of four species of Stegastes (S. pictus, S. fuscus, S. variabilis and S. leucostictus) found along the coast of Brazil. Stegastes pictus had a chromosomal number of 2n = 48 (14m+28sm+2st+4a, fundamental number (FN) = 92), S. fuscus had 2n = 48 (20m+22sm+6a, FN = 90), S. variabilis had 2n = 48 (18m+22sm+8a, FN = 88), and S. leucostictus had 2n = 48, (18m+22sm+8a, FN = 88). The nucleolar organizing regions were single and homologous in all of the species, and were located in the interstitial region on the short arm of the first submetacentric pair. The heterochromatin segments were reduced in size and were distributed conservatively over the centromeric and pericentromeric regions of most of the chromosomes. The marked divergence in the number of chromosomal arms, compared to other Perciformes (2n = 48, FN = 48), indicated that varying degrees of multiple pericentric inversions had occurred during the karyotypic evolution of the Pomacentridae. Subtle karyotypic differences between S. variabilis and S. leucostictus suggested a recent divergence or that their karyotypes were less susceptible to changes. These results indicate that cytogenetic analyses could provide important complementary data for the characterization of populations and species of Stegastes and damselfishes in general.