Objective Develop the identification method for puffer fish species and explorethe applicability of cytochrome oxidase subunit Ⅰ(COI) and cytochrome b(cytb) gene in species identification of Takifugu and Lagocephalus. MethodsCOI and cytb sequences of Takifugu and Lagocephalus were amplified and sequenced. Phylogenetic tree was built by MEGA 6.0 to determine the species of puffer fish. Several poisoning samples of roasted fish fillet were tested by the established method. Results Total 57 samples were classified into 9 species. Phylogenetic tree which were built based on COI and cytb gene could effectively distinguish most species except Lagocephalus spadiceus and Lagocephalus gloveri, Takifugu obscures and Takifugu coronoidus respectively. Lagocephalus lunaris were detected from poisoning samples. Conclusion DNA barcode method could effectively identify puffer fish and make up the defects in morphological identification.