Pyrazinamide (PZA) has remained a keystone of tuberculosis (TB) therapy, and it possesses high imperative sterilizing action that can facilitate reduction in the present chemotherapy regimen. The combination of PZA works both with first- and second-line TB drugs, notably fluoroquinolones, clofazimine, bedaquiline, delamanid and pretomanid. Pyrazinamide inhibits various targets that are involved in different cellular processes like energy production (pncA), trans-translation (rpsA) and pantothenate/coenzyme A (panD) which are required for persistence of the pathogen. It is well known that pncA gene encoding pyrazinamidase is involved in the transition of PZA into the active form of pyrazinoic acid, which implies that mutation in the pncA gene can develop PZA resistance in Mycobacterium tuberculosis (M. tuberculosis) strain leading to a major clinical and public health concern. Therefore, it is very crucial to understand its resistance mechanism and to detect it precisely to help in the management of the disease. Scope of this review is to have a deep understanding of molecular mechanism of PZA resistance with its multiple targets which would help study the association of mutations and its resistance in M. tuberculosis. This will in turn help learn about the resistance of PZA and develop more accurate molecular diagnostic tool for drug-resistant TB in future TB therapy.