Background and Objective: Drug resistance to tuberculosis is continuously increasing and is a significant threat to tuberculosis control programs because afew effective drugs are present against Mycobacterium tuberculosis. Although isoniazid (INH) is the most effective drug against tuberculosis, resistance to this drug also develops readily. Mutations in katG, specially the Ser315Thr substitution, are responsible for isoniazid resistance in a large proportion of patients with tuberculosis. However, the frequency of the katG Ser315Thr substitution varies among population samples. This study provided molecular characterization of isoniazid resistance of M. tuberculosis strains and extended our knowledge about molecular basis of M. tuberculosis drug resistance that is widely applicable for rapid drug resistance detection. Materials and Methods: Using 1% proportional method, the sensitivity of 126 strains isolated from patients in Isfahan and Tehran to isoniazid was determined. The katG mutations in codon 315 associated with isoniazid resistance among isoniazid resistant isolates was determined by PCR-RFLP. In this way, 355 bp PCR products were digested by MspI. Results: Out of 126 isolates of M. tuberculosis, 32 (25.4%) strains were determined as INH resistant. Resistance rate was 22.6% (19 strains) in Isfahan and 31% (13 strains) in Tehran. Overall, 72% of isoniazid-resistant isolates could be identified by analysis of just katG 315 loci. Conclusion: The PCR-RFLP using MspI restriction enzyme that detects katG Ser315Thr substitution could be identified in 72% of isoniazid-resistant strains. Elucidation of the molecular characterization of isoniazid resistance in M. tuberculosis has led to the development of different genotypic approaches to the rapid detection of isoniazid resistant in clinical isolates.