BACKGROUND: The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in beta subunit of RNA polymerase is main mechanism of resistance. METHOD: In this study, rpoB gene for the ~3 subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. RESULTS: All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S53 IL) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not comfirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513-515 were new identified mutations for the first time. CONCLUSION: Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.