ObjectiveTo optimize and establish a multiplex polymerase chain reaction （PCR） system to simultaneously identify Ziziphi Spinosae Semen （ZSS），Hovenia acerba semen （HAS），and Ziziphi Mauritianae Semen （ZMS）， etermine their content to solve the problem of adulteration of ZSS pieces and its preparations. MethodAfter the analysis and comparison of the internal transcribed spacer （ITS） sequence differences of ZSS and its adulterants，specific single nucleotide polymorphism （SNP） sites were found，and specific primers for identification were designed. The samples of ZSS，HAS， and ZMS from different sources were specifically amplified under the conditions of optimized annealing temperature，the number of cycles， and concentration of primers，as well as different polymerases and PCR systems after evaluation. Identification was carried out according to the size of specific amplification bands，and the lower limit of detection （LOD） and adulteration LOD were studied. ResultWhen the annealing temperature was 63 ℃ and the number of cycles was 23，549，169，389 bp specific bands were amplified from ZSS，HAS， and ZMS. The lower LOD of this method was 0.24 ng and 1.2 ng for ZSS and HAS， respectively. The adulteration LOD for ZSS，HAS， and ZMS was 0.5%，2%， and 2% respectively. ConclusionThe established multiplex allele-specific PCR identification method can accurately identify ZSS，HAS， and ZMS at the same time，which can provide a basis for solving the problem of adulteration of ZSS and references for controlling the quality，security， and clinical application of ZSS.