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Abstract: Sea cucumbers are regarded as traditional, delicious and luxurious seafood in Asia. Generally, it is difficult for species identification of sea cucumbers based on morphological characterization. In this study, Multiplex-PCR was developed to identify four sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, and Parastichopus californicus). After constructing the 692bp fragments of mitochondrial cytochrome oxidase I (COI) gene, four sets of species-specific primer were designed. The amplified lengths of fragments were 212bp for A. japonicus, 301bp for C. frondosa, 261bp for T. ananas and 358bp for P. californicus respectively, which could be obviously differentiated on DNA electrophoresis. The four sets of species-specific primer were mixed and applied to detect sea cucumber species simultaneously. Therefore, multiplex-PCR provided a convenient, useful, specific and academic technique to authenticate the sea cucumber species. This methodology can be very useful for traceability of sea cucumber species. [Copyright &y& Elsevier]