ABSTRACT: Genetic diversity among eight species of grouper, Epinephelus bleekeri, E. coioides, E. malabaricus, E. ongus, E. akaara, E. maculatus, E. merra and E. fuscoguttatus, was studied using six microsatellite loci, Em-01*, Em-03*, Em-07*, Em-08*, Em-10*, and CA-07*, with the aim of exploring the feasibility of using microsatellite data for species identification. The results showed high levels of genetic differentiation among species (FST = 0.4403 and RST = 0.4954). Species identification based on fixed allelic differences was possible between E. coioides, E. ongus, and E. fuscoguttatus at Em-01* and between E. fuscoguttatus and E. ongus at Em-08*. Private alleles were found in all species, except for E. ongus. Pairwise FST ranged 0.238–0.578 (P < 0.008 Bonferroni correction), and Nei's genetic distance ranged 0.433–2.710. Size homoplasy was observed at Em-03*157 allele, which was characterized by a T–C transition at the 119th nucleotide site of PCR products. The genetic assignment test unambiguously assigned each individual to the correct species. Thus, this test can be used for species identification of unknown individuals when the multilocus genotypes of the six microsatellite loci are available. The phylogenetic (neighbor-joining) tree, which was constructed based on the genetic distance matrix, separated the eight grouper species into two main groups.