BACKGROUND:Drug resistance is a leading concern in control of TB. Resistance against rifampin as one of the most important drugs in the treatment of Mycobacterium tuberculosisis caused by mutations in the 81-base pair region of the rpoBgene encoding the β-subunit of RNA polymerase. This study aimed to characterize the mutations in the rpoBgene associated with rifampin resistance among M. tuberculosis.METHODS: This study was conducted on referred samples of patients who did not respond to anti-TB treatment, in Tuberculosis Regional Reference Laboratory at Shariati Hospital. Drug susceptibility of M. tuberculosis isolates was surveyed using a proportional method on LJ medium. The isolates with resistant to rifampin were reconfirmed and then the rpoBgene was amplified and sequenced.RESULTS: Among 27 resistant cases, 8, 11 and 8 people were from Iran, Afghanistan, and Turkmenistan, respectively. In 26 out of 27 isolates, rpoBgene mutations were observed. The most prevalent mutations belonged to the codon 53.The most prevalent mutations belonged to the TCG (Ser) 531TTG (leu) with prevalence 51.8% (n=14), and GAC (Asp)516TAC (Tyr), CAC (His) 526GAC (Asp) and CAC (His) 526TAC(Tyr) mutations with prevalence 14.8%(n=4).Twenty-three isolates had just one mutation.CONCLUSION: The use of rpoBgene sequencing led to the lack of the need for growth of the organism in the culture medium, the direct use of clinical samples, reduction of biological risks and a detection about 96.3% of MDR TB cases lowering the cost of the treatment. KEYWORDS: Drug-resistant, Mycobacterium tuberculosis, rpoBgene, Rifampicin