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Aims: Pyrazinamide (PZA) is an important component of first-line tuberculosis (TB) treatment because of its distinctive capability to kill subpopulations of persister Mycobacterium tuberculosis (MTB). The significance of PZA can be understood by its inclusion in the most recent World Health Organization-recommended multidrug-resistant (MDR) TB regimen. Very little information is available about the prevalence of PZA-resistant TB from geographically distinct regions of high burden countries, including Khyber Pakhtunkhwa (KPK), Pakistan, because drug susceptibility testing (DST) of PZA is not regularly performed due to the complexity. In this study, we aimed to find the prevalence of PZA resistance in geographically distinct, Pashtun-dominant KPK Province of Pakistan and its correlation with other first- and second-line drug resistance. Materials and Methods: In this study, DST of PZA was performed through an automated BACTEC MGIT 960 system (BD Diagnostic Systems). The resistant samples were further subjected to DST of isoniazid (INH), rifampicin (RIF), ethambutol (EMB), streptomycin (SM), moxicillin (MOX), amikacin (AMK), ofloxacin (OFX), kanamycin (KM), and capreomycin (CAP). Results: Out of 1,075 MTB-positive isolates, 83 (7.7%) were found to be resistant to PZA. Among the PZA-resistant isolates, 76 (90–91.6%) and 67 (80–80.7%) were found to be resistant to INH and RIF, respectively, whereas 63 (76%) were resistant to both first-line drugs, INH and RIF (MDR-TB). The resistance level of EMB, OFX, and SM was also significantly high in PZA resistance, 35 (42%), 40 (48%), and 41 (49–50%) respectively. Conclusion: PZA resistance is significantly associated with other first- and second-line drug resistance. A significant number of PZA-resistant isolates are MDR cases. Therefore, DST of PZA should regularly be performed along with other drugs for better management of treatment of MDR and extensively drug resistant (XDR), to avoid side effects in patients. [ABSTRACT FROM AUTHOR]