The genus Epinephelus, which belongs to the family Serranidae, and the subfamily Epinephelinae, comprises about 100 species in all three major oceans. In this study, totally 33 Epinephelus species were collected in Taiwan, Australia and the West Pacific during January-December, 2012. The genetic data of the mitochondrial DNA COI gene were applied to investigate the evolutionary divergence (K2P). Furthermore, the phylogenetic tree of the neighbor-joining (NJ) method and the maximum parsimony (MP) method were also constructed. The results of evolutionary divergence revealed that E. chlorostigma and E. areolatus exhibited the minimum distance (0.004), while E. spilotoceps and E. quoyanus exhibited the maximum distance (0.210). According to the results of the phylogenetic tree, the genus Epinephelus had the following two clades, one contained mostly small- to medium-size “reticulated” groupers; while the other contained medium- to large-size groupers, such as E. lanceolatus. These results were similar to the traditional morphometric classification. These results revealed that mitochondrial COI gene is not only an effective “DNA barcoding” marker for species identification, but also a widely accepted marker for investigate the phylogenetic relationship among these commercially valuable groupers.