Abstract: Objective To investigate polymorphism of rpoB gene of Brucella melitensis strains isolated in China. Methods Species/biovars of B.melitensis stranis were identified by AMOS-PCR. Then,specific primers were designed according to four polymorphic loci of Brucella rpoB gene,four diversity loci of rpoB gene of 299 B.melitensis stranisincluding B.melitensis biovar 1(38),B.melitensis biovar 2(19),B.melitensis biovar 3(242)were amplified,and sequencing results of PCR product were compared with rpoB gene of standard strains of B.melitensis. Results Analysis of rpoB gene of 299 B.melitensis strains revealed that 169 strains were rpoB-2 variants genotype,103 were rpoB-2 genotype,24 strains exhibited new rpoB genotype(named as rpoB-4),3 strains of rpoB-1 genotype. Conclusion RpoB gene of B.melitensis from China has higher genetic polymorphism,rpoB gene can be used as a target gene for molecular epidemiological investigation of B.melitensis.