In this study, a multiplex PCR assay was developed for the rapid and accurate identification of three Chionoecetes species (Chionoecetes bairdi, C. opilio, and C. japonicus) available in global seafood markets. The morphological similarity between imported female C. bairdi from Japan and native C. opilio in Korea complicates visual discrimination and raises concerns over potential mislabeling. To address this issue, mitochondrial cytochrome c oxidase subunit I (COI) gene sequences were analyzed to identify species-specific SNPs, and primers were designed accordingly. Singleplex PCR confirmed species-specific amplification among the three target species, and the optimal annealing temperature was determined. The multiplex PCR simultaneously amplified distinct fragments of 598 bp (C. bairdi), 401 bp (C. opilio), and 194 bp (C. japonicus), with no nonspecific amplification or primer–dimer formation. Sensitivity testing revealed a detection limit of 0.01 ng/µL for all three species, defined as the lowest DNA concentration at which species-specific bands were consistently observed in at least two out of three replicates. These results demonstrate that the developed multiplex PCR is a reliable, rapid, and cost-effective tool for accurate species identification, supporting sustainable resource management, preventing seafood fraud, and ensuring safe distribution in both Korea and global seafood markets.

Reliable identification of seafood species is critical for fisheries management and product authentication, especially when morphological characteristics are lost during processing. In this study, a multiplex PCR system was developed to distinguish seven cuttlefish species (six Sepia spp. and Sepiella inermis) commercially distributed in the Korean seafood market. Species identity was first confirmed by amplifying a mitochondrial cytochrome c oxidase subunit I (COI) fragment (~658 bp) using universal primers (LCO1490/HCO2198), showing 99–100% sequence similarity to corresponding GenBank reference sequences. Analysis of genetic variation based on a 530 bp aligned region demonstrated complete interspecific differentiation without shared haplotypes among species. The number of haplotypes per species ranged from 5 to 21, with haplotype diversity values between 0.667 and 1.000. An extended COI fragment (~1200 bp) was further analyzed to identify diagnostic interspecific variation for marker development. Seven diagnostic single-nucleotide polymorphism (SNP) sites were identified and used to design species-specific forward primers with diagnostic nucleotides positioned at the 3′ termini. Distinct amplicons (220–1099 bp) were generated and clearly resolved by agarose gel electrophoresis. Because simultaneous amplification of all seven primer pairs reduced amplification efficiency, the assay was divided into two multiplex sets. Under optimized conditions (56 °C), each species produced a single expected band without cross-amplification. This multiplex PCR system provides a rapid and sequencing-free approach for reliable species discrimination and can be effectively applied to fisheries monitoring and seafood authentication in commercial supply chains.