Abstract: Objective　The study was conducted to distinguish the six species of Brucella in a single system using multiplex polymerase chain reaction(PCR). Methods　Five pairs of primers were designed according to the gene differences among the six species of Brucella. Mixture?鄄system based PCR was then conducted to identify the strains. Standard Brucella strains were used to establish the process and optimize experimental conditions. Wild strains were cloned for cross verification. Results　Multiplex PCR was capable of identifying each species of Brucella except for B. ovis. Conclusion Multiplex PCR can serve as a quick, accurate and safe supplementary method for the identification of Brucella.