Background: Early detection and suitable drug admission are the first attempts to control of TB and MDR-TB. In this study, PCR-RFLP method was used for rapid detection of Mycobacterium tuberculosis and resistance to isoniazid. Materials and Methods: In the present study, 87clinical isolates of MTB were used in a PCR-RFLP method. The RFLP-PCR assay by specific primer was used for two purposes In the first, PCR of katG gene for diagnosis of bacteria and in the second step, PCR product by endonuclease enzyme in a RFLP reaction produced a pattern in electrophoresis for mutation detection in katG315. Sequencing method was used for confirmation of RFLP results. Results: The study showed that all studied strains produced band 620bp that confirmed MTB. From the 46 resistant strains to isoniazid, the RFLP-PCR method showed that 44 strains had mutations in the katG gene Ser315Thr. The 41 susceptible strains had not any mutation at the codon. Results of sequencing were confirmed results of the molecular method. Sensitivity of RFLP-PCR test was 95.6% (95% CI :0.85-0 .98) and specificity was 100% (95% CI :0.91-1 .0). Conclusion: RFLP-PCR test is a good tool for simultaneous diagnosis and detection of katG315 mutation in clinical isolates of MTB.