Abstract: Objective：To characterize rpoB mutations in rifampinresistant Mycobacterium tuberculosis clinical isolates in Wuhan district, China. Methods：The rpoBgene fragment including 81 bp of RRDR (rifampin resistance determination region) was analyzed following sequencing. Of the 76 Mycobacterium tuberculosisstrains, 56 strains were resistant to rifampin, and 20 strains were rifampin-susceptible. Results：Among the rifampin-resistant isolates 92.9% (52/56) carried mutations in the amplified region of rpoB gene, involving 18 different types of mutations in 10 codons. The mutations at codon 531 and 526 were more frequently observed with the rate of 57.7% (30/52) and 19.2% (10/52), respectively. The combined mutations rate was identified as 13.5%（7/52）. A new mutation type 509 (AGC→AGA) was found. The CAG deletion type of 517 which was rarely observed in China was identified. Conclusion：The mutations in rpoB gene are widely existed in rifampin-resistant Mycobacterium tuberculosis clinical isolates in Wuhan district. New mutation type may be found in succession.