Differentiation of M. tuberculosis complex organisms were assigned to one of three genotypic groups based on the combinations of polymorphisms at katG codon 463 and gyrA codon 95. Early identification of strains belonging to any particular group is very important. This study was planned to identify major genetic groups of clinically isolated Mycobacterium tuberculosis. Materials and Methods: In this cross sectional study 33 sputum samples were collected from tuberculosis patients of the Markazi

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... province. DNA purification from isolated samples was performed by Chelex 100. Identification of isolates was confirmed by detection of katG gene and the mutation in KatG463 by using PCR method and RFLP respectively. Finally 620-bp of katG gene and 194-bp of gyrA gene purified from PCR product were sequenced. Results: Amplification of 620-bp fragment of katG gene was a good way to confirm the detection of bacteria as a molecular approach. Results of sequencing codon GyrA95 in combination by results of PCR-RFLP determined type of the major genetic group (MGG). Therefore it showed that among the 33 Mycobacterium tuberculosis isolates 12 samples were MGG 1, 15 Samples were MGG2 and 6 samples were MGG 3. Results revealed that MGG 2 was dominant form of M. tuberculosis strains of Markazi province by frequency of 45.5%. Conclusion: Based on the results of this study MGG2 occurrence was more frequent among clinical strains in Markazi province that its accordance with susceptibility of these strains to conventional antibiotics is notable. In this study, three applicable benefits from the test as: MGG typing, molecular detection of M. tuberculosis and bacterial resistance to Isoniazid were proven.