Objective：Examine the variations in medication resistance between rifampicin (RIF) and rifabutin (RFB) of MTB with rpoB gene mutationsbut but phenotype is sensitive . Methods：During January 2022 to December 2024, The sputum or bronchoalveolar lavage fluid samples from tuberculosis patients in our institution were collected and sent to the bacteriology laboratory for MTB culture and Xpert testing. Subsequently, rpoB gene sequencing was performed on molecularly resistant strains, and their drug susceptibility results were analyzed in the same time. Finally, We selected all RIF-sensitive and low-level resistant strains and some high-level resistant strains Among rpoB gene mutant strains, and determinedand the MICs of RIF and RFB using the concentration gradient method. Results：A total of 298 strains with both molecular and phenotypic drug susceptibility positive were collected. The molecular drug susceptibility method exhibited a sensitivity of 93.3%(95%confidence interval 87.7%-99.0%)for RIF resistance，and a specificity of 94.6%(95%confidence interval 91.4%-97.3%)，resulting in an overall accuracy rate of 94.3%.The 82 molecularly resistant strains，which were found to have rpoB gene mutations in 79 strains(94%).531, 526, and 511 residues were The common mutation sites . The results of the agar solid drug susceptibility testing indicated that the S531L，H526Y，H526R，H526Q，and D516V sites exhibited high resistance to RIF， while the S512G，S522L，and L533P sites demonstrated moderate resistance. In contrast，the H526L，D516Y，L511P，and P483L sites were found to be sensitive to RIF The rpoB mutant strains were used to test the MIC of RIF and RFB.Only H526L and L511P mutant forms were sensitive to both RIF and RFB. RIF-resistant but RFB-sensitive mutant forms included: one strain with a base mutation rate of 20.34% of S531L, L511P combined with D526Q double mutation, H526D, D516Y, S512G, S522L, L533P, and P483L. For all rpoB mutant strains, the log-transformed RIF MIC (Log 2RIF-MIC) value [3.5 (1.0, 4.0)] was significantly higher than the log-transformed RFB MIC (Log 2RFB-MIC) value [2.0 (-3.0, 3.75)] (Z = -4.481, P < 0.001). Conclusions：Studying the differences in RIF and RFB resistance among MTB strains with different rpoB gene mutation sites might be helpful in creating diagnostic tools and offering suggestions for stratified treatment for drug-resistant tuberculosis.