Pyrazinamide (PZA) has been in use for almost 50 years as a first-line drug for short-course chemotherapy against Mycobacterium tuberculosis. In this study, PCR mediated automated DNA sequencing is used to check the prevalence of PZA resistance among treatment failure cases of pulmonary tuberculosis. Out of 50 clinical isolates examined, 39 had mutations in the pncA gene that encodes Pyrazinamidase, an enzyme required to activate PZA. Of these, 31 (79.5%) were localized to three regions of pncA. We found two isolates with hitherto unreported mutation at amino acid 26 (Ala -> Gly) of pncA.