To study the phylogenetic relationships of the genus Gymnothorax (moray eels) distributed in South China Sea, polymerase chain reactions were performed, and the amplification products were sequenced by cloning into the PMD18T-vector (TaKaRa). The entire gene sequences encoding cytochrome b (1140 bp) for 16 Gymnothorax (G. flavimarginatus, G. meleagris, G. undulates, G. reticularis, G. reevesi, G. melanospilus, G. rueppeliae, G. javanicus, G. chilospilus, G. pseudothyrsoideus, G. fimbriatus, G. hepaticus, G. berndti, G. curostus, G. favagineus, and G. margaritophorus) were obtained. Four additional Gymnothorax sequences from GenBank were also included. The nucleotide composition, genetic distances, and base substitution saturation analysis were calculated using the MEGA 5.0 Software. Phylogenetic analysis was performed using maximum-parsimony, maximum-likelihood (ML), and neighbor-joining (NJ). The results were as follows: 1) base-substitution saturation analysis suggested that both in third codon positions, and the full-length cytochrome b data set, Ts are not saturated, but Tv substitutions may be saturated, 2) the genus Gymnothorax, native to the South China Sea, is divided into four distinct clades, with two clades in the NJ and ML trees, and 3) according to our experimental data, G. melanospilus (Bleeker, 1855) and G. favagineus (Bloch and Schneider, 1801) are the same species.