Abstract: Objective To analyze the characteristics of rpoB gene mutation in rifampicin-resistant mycobacterium tuberculosis isolate from Yunnan province. Methods This study selected 102 mycobacterium tuberculosis isolates and 1 strain of H37Rv standard mycobacterium tuberculosis. The fragments of rpoB gene including rifampicin resistance determining region were amplified by polymerase chain reaction (PCR), followed by sequencing. Results were analyzed by MEGA 5 software. Results The coincidence rate of rifampicin-resistant genotype and phenotype was 92.00%(69/75). There were 11 species of mutations which affect amino acid sequence. All mutations were point mutations, among which, 92.96% were single codon mutations and 7.04% were combination mutations. The top 3 codon mutations were rpoB531 (43.66%), rpoB526 (26.76%) and rpoB516 (9.86%), accounting for 80.28% of all mutations tested. There was no difference in rpoB gene mutations in clinical isolates from new patients and retreatment patients. There was a difference in the composition of rpoB mutations in MDR-TB and rifampicin resistant. Conclusions The detection of rpoB mutations in Yunnan Province can be used as an important indicator for rifampicin-resistant screening. The rpoB gene mutations may not be affected by previous administration of rifampicin and the duration of administration, while isoniazid may affect the molecular mechanism of rpoB gene mutations in Mycobacterium tuberculosis.