Abstract: To analyze the molecular systematic and phylogenetic relationships of Epinephelus fish, mitochondrial COⅠ and nuclear TMO-4C4 gene sequences from 142 samples of 35 Epinephelus species collected from Western Pacific coastal areas were determined. Inter-species and intra-species genetic distances were calculated using MEGA 7.0 and the molecular phylogenetic trees were constructed using maximum likelihood and Bayesian inference methods. The availability of using COⅠ sequences as DNA barcodes to identify Epinephelus species were also investigated. The results showed that the homologous COⅠ sequences of 35 species were 739 bp, encoding 243 amino acids and the homologous TMO-4C4 sequences were 486 bp, encoding 162 amino acids. In the COⅠ gene sequences, the inter-species genetic distances among 35 groupers were between 0.030—0.202 and the average distance was 0.143, which was greater than the minimum species identification value 0.200 suggested by Hebert. The genetic distances of intra-species were between 0.000—0.008, with an average of 0.003. The average inter-species value (0.143) was 48 times larger than that of intra-species one (0.003), indicating that COⅠ may be used as an effective barcode gene for accurate identification in Epinephelus species. In the molecular phylogenetic trees constructed by COⅠ and TMO-4C4 sequences, 35 Epinephelus species formed two major clades. Clade Ⅰ comprised of 22 species including Epinephelus cyanopodus, Epinephelus multinotatus, etc; Clade Ⅱ comprised of 13 species including Epinephelus morrhua, Epinephelus epistictus etc. Certain pairs of groupers like E. cyanopodus/E. multinotatus, E. morrhua/E. epistictus, Epinephelus bilobatus/Epinephelus maculates, Epinephelus coeruleopunctatus/Epinephelus corallicola, Epinephelus moara/Epinephelus bruneus, Epinephelus coioides/Epinephelus malabaricus were tightly clustered into sister species with high support values in the phylogenetic tree. Basing on the sequence divergences and phylogenetic analyses of the COⅠ and TMO-4C4 gene, great genetic differences were found between the species: E. moara/E. bruneus and E. coioides/E. malabaricus which were confronted with synonymous controversies. This results supported the viewpoints of recent morphological studies that E. moara/E. bruneus and E. coioides/E. malabaricus weredistinct species and should not be placed as synonym. This study provides molecular evidence to clarify the classificational controversy and to accurately identify Epinephelus species.