Abstract Background: Tuberculosis (TB) is a major public health Problem in the 21st Century. Pyrazinamide (PZA) is a drug used in the treatment of pulmonary TB and is used in short-course treatment in combination with rifampin, isoniazid and ethambutol. Drug resistance to TB is of major public health concern. More threat is on PZA resistance which is a key drug in the treatment of both drug-susceptible and drug-resistant TB. Aim: The aim of this study was to determine the prevalence and characterization of pyrazinamide resistance of Mycobacterium tuberculosis from TB isolates. Methods: Seventy nine archived TB isolates from patients with pulmonary TB were re-cultured into L-J and MGIT tubes. The colonies from L-J were used to extract DNA and perform HRM and DNA sequencing whereas colonies from MGIT were used to set up MGIT PZA susceptibility test. The results of drug-resistance in the pncA gene determined by HRM were compared to the phenotypic results determined by MGIT. Results: The prevalence of PZA resistance was found to be 25.3% by MGIT PZA and 40.5% by HRM. Characterization of HRM for 32 resistant isolates revealed 1(3%) for pncA1, 4 (12.5%) for pncA2, 7(21.9%) for pncA3, 15 (46.9) for pncA5, 19 (59.4%) for pncA6, 4(12.5%) for pncA7 3(9.4%) for pncA8 and 6 (18.8%) for, pncA9. DNA sequencing depicted 7 (31.8%) to be mutants of which, 6 (85.7%) possessed mutation at Ser65Ser and 1 (14.3%) isolate with mutation at Leu35Arg. Conclusion: We conclude that phenotypic MGIT PZA has to be use in parallel molecular methods in routine diagnosis so as to detect false susceptible and false resistant MGIT PZA results. Keywords: tuberculosis, multidrug-resistant tuberculosis, Tanzania, prevalence, characterization