Dolphinfishes (Coryphaenidae) are pelagic predators distributed throughout all tropical and subtropical oceans and are very important for commercial, traditional, and sport fishing. This small family contains the Coryphaena hippurus and Coryphaena equiselis species whose chromosomal aspects remain unknown, despite recent advances in cytogenetic data assimilation for Perciformes. In this study, both species were cytogenetically analyzed using different staining techniques (C-, Ag-, and CMA3 banding) and fluorescence in situ hybridization, to detect 18S rDNA and 5S rDNA. C. hippurus females exhibit 2n = 48 chromosomes, with 2m+4sm+42a (NF = 54). In C. equiselis, where both sexes could be analyzed, females displayed 2n = 48 chromosomes (2m+6sm+40a) and males exhibited 2n = 47 chromosomes (3m+6sm+38a) (NF = 56), indicating the presence of X1X1X2X2/X1X2Y multiple sex chromosomes. Sex-chromosome systems are rare in Perciformes, with this study demonstrating the first occurrence in a marine pelagic species. It remains unknown as to whether this system extends to other populations; however, these data are important with respect to evolutionary, phylogenetic, and speciation issues, as well as for elucidating the genesis of this unique sex system.