Genus Epinephelus (Perciformes, Epinephelidae), commonly known as groupers, are usually difficult in species identification for the lack and/or change of morphological specialization. In this study, molecular cytogenetic analyses were firstly performed to identify the closely related species Epinephelus bruneus and E. moara in this genus. The species-specific differences of both fish species showed in karyotype, chromosomal distribution of nucleolar organizer regions (NORs) and localization of 18S rDNA. The heterochromatin (interstitial C-bands) and distribution pattern of telomere (TTAGGG) n in E. bruneus revealed the chromosomal rearrangements and different karyotypic evolutionary characteristics compared to those in E. moara. The cytogenetic data suggested that the lineages of E. bruneus and E. moara were recently derived within the genus Epinephelus, and E. moara exhibited more plesiomorphic features than E. bruneus. All results confirmed that E. moara, which has long been considered a synonym of E. bruneus, is a distinct species in the family Epinephelidae. In addition, molecular cytogenetic analyses are useful in species differentiation and phylogenetic reconstruction in groupers.