Objective：To examine the variations in drug resistance between rifampicin（RIF）and rifabutin（RFB）in Mycobacterium tuberculosis（MTB）with rpoB gene mutations but phenotypically sensitive. Methods：Sputum or bronchoalveolar lavage fluid samples from pulmonary tuberculosis patients at the Affiliated Nanjing Hospital of Nanjing University of Chinese Medicine from January 2022 to December 2024 were collected. These samples underwent MTB culture，Xpert molecular drug susceptibility testing（DST），and rpoB gene sequencing. Strains with rpoB mutations that were phenotypically sensitive，showed low-level resistance，or selected cases of highevel resistance to RIF were screened. The minimum inhibitory concentrations（MIC）of RIF and RFB were determined using the concentration gradient method. Results：A total of 298 strains were included，with both molecular and phenotypic DST being positive. The molecular DST showed a sensitivity of 93.3% and a specificity of 94.6% for detecting RIF resistance，with an overall accuracy of 94.3%. Among 82 molecularly resistant strains，rpoB gene mutations were detected in 96.3%，with common mutation sites at codons 531，526，and 511. Agar-based phenotypic DST results showed that S531L，H526Y，H526R，H526Q，and D516V mutations conferred high -level resistance to RIF；S512G，S522L，and L533P conferred low -level resistance；while H526L，D516Y，L511P，and P483L mutations were phenotypically sensitive to RIF. MIC testing revealed that only H526L and L511P mutations were sensitive to both drugs. In contrast，strains with mutations including S531L，L511P combined with H526Q，H526D，D516Y，S512G，S522L，L533P，and P483L were resistant to RIF but remained sensitive to RFB. Overall，the log-transformed RIF MIC（log2 RIF-MIC）value[3.50（1.00， 4.00）]for rpoB mutant strains was significantly higher than the log-transformed RFB MIC（log2 RFB-MIC）value[2.00（-3.00，3.75）] （Z=-4.481，P ＜ 0.001）. Conclusion：This study demonstrates that different rpoB mutation sites lead to varying levels of resistance to RIF and RFB in MTB. Compared to RIF，RFB exhibits lower MIC values and potential clinical effectiveness against various rpoB mutant strains（e.g.,L511P，H526L，D516Y，S522L，etc.）. Precise analysis of rpoB mutation sites and evaluation of their susceptibility to RFB may offer a new strategy to address the clinical dilemma of“molecular resistance-phenotypic sensitivity”and provide a basis for formulating individualized，stratified treatment plans using rifamycin drugs.