In this comparative study, all the five Brucella strains were morphologically, serologically and biochemically identified using media different stains and antibiotics. Multiplex PCR was used to differentiate between the Brucella strains and Yersinia enterocolitica O: 9. five primer sets were designed to the most specific variable regions of the different Brucella species. The results of multiplex PCR were very successful and accurate in terms of characterization and typing of the Brucella vaccine, reference and wild Brucella strains from Yersinia which give false positive while using tests based on anti-LPS antibodies but with PCR for DNA of Yersinia give negative. The molecular typing of the Brucella strains by multiplex PCR had several advantages over the use of the conventional methods being very fast, precise, easier, more sensitive, and economic and could be applied on minimal sample preparation. The development of this PCR method is the first step toward the development of a novel kit for the molecular identification of Brucella strains from other Gram- negative bacteria.