Background: The emergence of drug-resistant strains of Mycobacterium tuberculosis (MTB) is an increasing problem in developed and developing countries. The aims of the present study were to identify various types of mutations in katG region from 28 MDR strains isolated from sputum of tuberculosis patients. Materials and Methods: Twenty-eight rifampin-resistant strains isolated from sputum of patients with active pulmonary tuberculosis were obtained from various geographic regions of Iran. Drug susceptibility was determined by using the BACTEC system. DNA extraction, standard PCR identification, katG gene amplification, DNA sequencing and analysis were done. Results: There was no mutation in 2 strains. In 20 strains, mutation was shown to be in codon 315. Three types of mutations were detected consisting of AGC→ACC (Ser→Thr) (80%), AGC→AGG (Ser→Arg) (5%) and AGC→AAC(Ser→Asn) (15%).Furthermore, one type of mutation was seen in codons 311, 299, and 323. Twelve strains showed one mutation in codon 315 (46%), 7 strains 2 mutations (27%), 5 isolate 3 mutations (19%) and in 2 strains 4 mutations (8%) were observed in different codons. Nine silent mutations was demonstrated in codon 311(GAC→TAC ). Conclusion: This research demonstrated that mutations were mostly seen in codons 315 and 299 indicating resistance to isoniazide. (Tanaffos 2006; 5(1):31- 36)