A recent taxonomic review has revealed that three species of Ammodytes (Ammodytes japonicus, Ammodytes heian, and Ammodytes hexapterus) are distributed in Japanese waters. Species identification using morphological characters is difficult due to their similar appearance. A rapid and convenient method for identifying the three species of Ammodytes from Japan was developed via a multiplex haplotype-specific (MHS)-PCR of the mitochondrial COI region. Two primers designed for A. japonicus and A. hexapterus yielded species-specific amplification products in different sizes (ca. 500 bp and 300 bp, respectively). Together with a pair of universal primers for fish, about 1,500 bp can be amplified by MHS-PCR, which allows the robust identification of the three species of Ammodytes.