This study was planned to verify the resistance frequency of Ofloxacin (OFX) against Mycobacterium tuberculosis by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique and sequencing. Total 366 clinical samples of suspected TB patients were collected from various localities of central Punjab. All of them were found positive by ZN (Zeihl-Nelsen) staining method. Among them, 108 (29.5%) were found negative and 258 (70.5%) positive on PCR based study. The cases not responding to ATT were further characterized by proportion method and by PCR-RFLP to establish the drug resistance. Selected drug resistant case were further sequenced to confirm the results of amplified RFLP. The results showed that out of 118 drug resistant cases, 06 (5.08%), 03 (2.54%) were found resistant to OFX by drug susceptibility testing and PCR-RFLP respectively. The two strains were selected for sequencing procedure. The strain-79 showed point mutation at four points, at codon 70, 71, 76 and 78. The sequence of strain- 81 showed mutation at codon 95.PCR-RFLP is a useful molecular technique for the rapid detection of mutations and may be used to diagnose drug resistance but it should be confirmed by sequencing before starting 2(nd) and 3(rd) generation treatment because the restriction site is the cornerstone of PCR-RFLP and mutation may be occurring elsewhere.