The systematic position of the amphioxus species with dextral gonads distributed in the southeast Pacific and Indian Oceans has remained to be clarified due to the adoption of different names by different researchers. Mitochondrial (mt) DNA is generally considered to be a powerful molecular marker in taxonomic studies. For a reliable systematics of the amphioxi collected from the South China Sea, we sequenced the complete mtDNA from a single specimen and compared it with those of the other eight amphioxus species. The present mtDNA genome contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, with the same gene order as those in Branchiostoma and Epigonichthys, which, however, is different from that in Asymmetron. Based on our morphological data (including measurements of some characters) and the features of the mt genome, together with the distribution records of the dextral-gonad amphioxi, we conclude that the present mtDNA sequence most likely represents that of E. cultellus.