Ornate threadfin bream (Nemipterus hexodon) is an economically important fishery species in Southeast Asia. In Thailand, N. hexodon decreased dramatically due to overexploitation for commercial purposes. To construct an effective sustainable management plan, genetic information is necessary. Thus, in our study, the population genetic structure and demographic history of N. hexodon were investigated using 419 bp of the mitochondrial DNA sequence in cytochrome oxidase subunit I gene (mtDNA COI). A total of 142 samples was collected from nine localities in the Gulf of Thailand (Chonburi, Samut Songkhram, Surat Thani, Nakhon Si Thammarat, Songkhla), and the Andaman Sea (Satun, Trang, Krabi, Phang Nga). Fourteen polymorphic sites defined 18 haplotypes, revealing a high haplotype diversity and low nucleotide diversity among nine localities. The analysis of molecular variance (AMOVA) analysis, pairwise F ST , and minimum spanning network result revealed that the genetic structure of N. hexodon was separated into two populations: the Gulf of Thailand and the Andaman Sea population. The genetic structure of N. hexodon can be explained by a disruption of gene flow from the geographic barrier and the Pleistocene isolation of the marine basin hypothesis. Neutrality tests, Bayesian skyline analysis, mismatch distribution, and the estimated values of population growth suggested that N. hexodon had experienced a population expansion. The genetic information would certainly help us gain insight into the population genetic structure of N. hexodon living on the coast of Thailand.