Abstract： Objective To characterize rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis (M.tuberculosis) in Zhejiang Province.Methods A total of 188 clinical isolates of M.tuberculosis from 188 patients with pulmonary tuberculosis in Tongde Hospital of Zhejiang Province and Integrated Chinese and Western Medicine Hospital of Zhejiang Province were collected.Conventional drug resistance analysis was performed and the mutation of rpoB gene was detected by PCR-based DNA sequencing.The association between gene mutations in rifampin-resistance determining region of M.tuberculosis and clinical resistance was analyzed.Results Fifty-seven out of 188 isolates (30.3％) were drug-resistant strains,including 18 isolates (9.6％) with single-resistance to rifampin,28 isolates (14.9％) with single-resistance to other anti-tuberculosis drugs (10 to isoniazid,12 to streptomycin and 6 to ethambutol),and 11 isolates (5.9％) with multi-drug-resistance (rifampin plus one or more drugs of isoniazid,streptomycin and ethambutol).Among 29 rifampin-resistant strains,rpoB gene mutation existed in 27 strains (93.1％),and the most frequently mutated sites were codons 526 (55.6％,16/27),513 (22.2％,5/27),531 (14.8 ％,4/27)) and 529 (7.4％,2/27).Among 28 strains which were resistant to other anti-tuberculosis drugs,rpoB mutations existed in 4 strains (14.3％),and the mutated sites were codons 526 (2 strains) and 513 (2 strains).All 13 sensitive isolates had no mutation in rpoB gene.Conclusion Rifampin resistance in M.tuberculosis is closely correlated with rpoB gene mutations in Zhejiang province,and the most frequent sites of mutation are at codons 526 and 513.