From mid-December to late January, schools of mature gray mullet (Mugil cephalus) migrate southward along the coastal waters of China to Taiwan for spawning. It has been proposed that there is no genetic differentiation of gray mullet in the coastal waters of Taiwan. To test this hypothesis, complete cytochrome b (cyt b) DNA sequences of 98 individuals of gray mullet, two individuals of Liza macrolepis, and three individuals of L. affinis were amplified by polymerase chain reaction and sequenced. Phylogenetic trees reconstructed with the Bayesian, maximum likelihood, maximum parsimony, and neighbor-joining methods all support the existence of three monophyletic groups (denoted as groups 1, 2, and 3), with net evolutionary divergences (p-distances) between groups ranging from 5.1% to 6.6%. To estimate the relative abundance of each group, a PCR-RFLP method was developed to examine 600 juveniles collected from October 2006 to March 2007. Groups 1, 2, and 3 comprised 85%, 3%, and 12% of the samples, respectively. Juveniles of groups 1 and 3 could be found as early as November, but juveniles of group 2 were not found until February. Based on the dates of specimen collection and phylogenetic analyses, we propose that groups 1 and 2 are migratory populations from China and Japan, respectively, whereas group 3 is a resident population in Taiwan.