Pyrazinamide (PZA) is an important anti-tuberculosis drug, which is active against semi-dormant bacilli and used as a component of first-line drugs and drug-resistant tuberculosis regimens. Mutations in pncA and its promoter region are main cause of PZA resistance. There are limited PZA susceptibility data as there is no routine drug susceptibility testing (DST) for PZA. This study was aimed to determine the proportion of PZA resistance among rifampicin-resistant tuberculosis patients and to identify mutations which are responsible for PZA resistance in pncA and its promoter region. Liquid-based DST was performed to detect PZA susceptibility on 192 culture positive rifampicin-resistant isolates collected from National Tuberculosis Reference Laboratory. Sequencing on pncA including its promoter region was performed and analysis was done on 157 isolates. Phenotypic PZA resistance was detected in 58.9% of isolates. Sixty-five different mutations were distributed in pncA or promoter region of 82 isolates. Sensitivity and specificity of pncA sequencing in detection of PZA resistance showed 89.8% and 95.6% respectively. High proportion of PZA resistance among rifampicin-resistant cases highlighted the need for effective treatment regimen development for PZA-resistant MDR-TB. It is also suggested that routine PZA susceptibility test should be incorporated to treatment monitoring regimen and National Drug Resistance surveys.Copyright © 2020. Published by Elsevier Ltd.