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Girella punctata and Girella leonina are economically important species found in the East Sea; along the southern coast of Korea; south of Hokkaido, Japan; around Taiwan; and in the East China sea. In Korea, these two species hold high value, particularly on Jeju Island. These species have similar appearances, and it is challenging to distinguish them, particularly during the seed period. We detected genetic differences in the mtDNA (COI gene) of G. punctata and G. leonina, which are morphologically indistinguishable, and developed species-specific genetic markers for their identification. In total, 16 and 4 haplotypes of the COI genes were obtained from G. punctata (n = 164) and G. leonina (n = 36), respectively. The haplotype diversity (Hd) and nucleotide diversity (Pi, %) of the COI were 0.359 and 0.054 for G. punctata and 0.560 and 0.078 for G. leonina, respectively. We designed a Girella species common primer (control) and species-specific primer sets (experimental) for the two species. Amplicon sizes of 991, 579, and 391 bp were obtained for common primers of the two Girella species G. punctata and G. leonina. To confirm multiple targets in a single reaction, multiplex PCR conditions were optimized to adjust its resolution and accuracy. The detection levels of the multiplex PCR were confirmed to be 0.01 ng/µL for the two Girella species. The multiplex PCR was not associated with cross-reactivity between G. punctata and G. leonina. This multiplex species-specific PCR method provides a simple and rapid technique for the identification of two Girella species, thus increasing the efficiency and quality of Girella species stock management and forensic identification to prevent species misidentification. [ABSTRACT FROM AUTHOR]