Abstract: Objective To explore the mutation characteristics of rifampin gene of Mycobacterium tuberculosis in patients with pulmonary tuberculosis, and analyze the drug resistance, so as to provide scientific basis for the prevention and treatment of drug-resistant tuberculosis. Methods Total 532 patients with pulmonary tuberculosis admitted to the Affiliated Hospital of Hangzhou Normal University from June 2016 to December 2018 were divided into drug-resistant strains(n=218) and sensitive(n=314) strains according to the drug resistance of Mycobacterium tuberculosis in sputum. The rpoB gene sequence and minimal inhibitory concentration(MIC) value were determined. Results Among the 218 resistant strains, 180 were single mutation resistant strains. The mutation codon with the highest rate of rpoB gene structure analysis was 526(13.2%) and 531(74.4%). Total 206(94.5%) resistant strains showed at least one mutation site, and all of them were located in the rifampicin resistant decision region(RRDR), and 12(5.5%) resistant strains were outside RRDR. Four resistant strains had synonymous mutation in RRDR and 4 resistant strains had synonymous mutation outside RRDR. There was no mutation in H37 Rv codon. Eight sensitive strains had synonymous mutations outside RRDR. The RIF MIC level of the single-mutated strain was(47.34±2.04) μg/mL, which was significantly lower than that of the mutant strain(118.24±3.95) μg/mL(t=107.636, P<0.001). There was no significant difference between the MIC level of single codon 526 and single codon 531 mutant strains(P>0.05). The MIC level of the multicodon mutant was(116.03±5.06) μg/mL higher than that of the single codon(47.08±1.31) μg/mL(t=85.530, P<0.001). Conclusion The mechanism of rifampicin resistance may be related to the mutations at 531 and 526 of the start of rpoB gene. The rpoB gene sequence can be used to predict the resistance and phenotype of rifampicin in Mycobacterium tuberculosis.