The study aims to establish a multiplex polymerase chain reaction (PCR) assay for identification of Mylopharyngodon piceus,Ctenopharyngodon idella,Hypophthalmichthys molitrix, and Aristichthys nobilis simultaneously. Based on the blast results of D-LOOP gene of M. piceus, COI gene of C. idella, 16SrRNA gene of H. molitrix and 12SrRNA gene of H. nobilis, four pairs of specific primers were designed to for the four fish different gene sequences. The identification method of four fish was established by single PCR and multiple PCR. The optimal conditions for PCR were explored by orthogonal experimental design L₉(3⁴). The results showed that the size of anticipated PCR products for the target gene of M. piceus, C. idella, H. molitrix and H. nobilis were 298、210、306 and 639 bp respectively. Under the optimized conditions, the method has a high specificity, and the 4 fish target gene in plasmid could be amplified simultaneously with clear bands at the amount of template lower to 50 pg. Furthermore, the method was stable by verifying in simulated examination and marketing examination. The study concluded that a rapid, specific and sensitive multiplex PCR method was established for identifying and discriminating fish products compositions from M. piceus, C. idella, H. molitrix and H. nobilis. [Chinese Fishery Quality and Standards,2021,11(1):11-17]