Background: Prevention and treatment of drug-resistant clones is important in guiding TB control strategies. The simultaneous rapid detection of the type of mutation conferring resistance and the genotype reflect the extent of drug resistant TB transmission within the communities.Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. Materials and Methods: Spoligotyping, IS6110- restriction fragment length polymorphism (RFLP) typing and sequencing of the rpoB gene were performed for 30 rifampin resistant M. tuberculosis isolates from patients referred to "Iranian National TB Laboratory" from 2006 to 2007. Results: Mutations in the RRDR of the rpoB gene were identified in 96.6% of rifampin-resistant isolates. The spoligotyping analysis identified one (3.3%) East African-Indian (EAI) family, 7 (23.3%) Haarlem family, 9 (30.0%) Beijing family and 12 (40.0%) Central Asia (CAS) family isolates. Sixty- six percent of CAS isolates carried a mutation in codon 516, 37% of Beijing isolates carried a mutation in codon 531 and 33% of Haarlem isolates carried a mutation in codon 526. Conclusion: Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampin in the Beijing and Haarlem families. (Tanaffos 2008; 7(2): 11-17)