Abstract: Asingle gene fragment and combined nucleotide sequences for two gene fragments of mitochondrial DNA (mtDNA), which were 16S ribosomal RNA (16S rRNA) and cytochrome oxidase c subunit 1 (COⅠ), were analyzed for three populations of Trichiurus lepturus (T. lepturus, designated by A, B, and C). The length of 16S rRNA gene of mtDNA and COⅠ was 1130 and 554 bp, respectively. These two gene fragments were combined together to form a gene fragment with the length of 1684 bp for the purpose of subsequent genetic diversity analysis. The average contents of nucleotides T, C, A, and G were 29.0%, 28.9%, 24.4% and 17.7%, respectively in the mtDNA COⅠgene, while those mean contents were 22.7%, 27.6%, 28.0% and 21.7% respectively in 16S rRNA gene. In this study, 43, 8, and 49 haplotypes were defined in 72 individuals by using single and combined gene fragments, in which haplotypes were shared among the mentioned populations. The diversities of haplotypes varied between 0.9766 and 0.9992, indicating that the abundance of haplotypes was within different populations. The nucleotide differences and the nucleotide diversity in each population were in the ranges of 5.111—9.024 and 0.0045—0.0076, respectively. Genetic distances among the populations varied from 0.0048 to 0.00084. These results indicated that genetic diversity in three populations of T. lepturus was considerable. The construction of phylogenetic trees based on Neighbour-Joining (NJ) method showed that several individuals in the same population were aggregated. The achieved findings also indicated that high frequency of gene transfer among different populations resulted in low genetic differentiation. Population A contained richer genetic background than that of populations B and C. The difference within different populations was higher compared to among various populations. It could be concluded that the three mentioned populations of T. lepturus had low genetic diversity based on single and combined mtDNA genes.