Pyrazinamide (PZA) is one of the first-line anti-tuberculous drugs used in the treatment of tuberculosis (TB). Considering the ability of PZA to shorten the treatment period from 9-12 months to six months by eliminating persistent bacilli, it appears to be an important cornerstone of TB therapy. While the main mechanism causing the PZA resistance is pncA mutations at a rate of 70-97%, it has been determined that rpsA and panD mutations can also cause resistance. In this study, we aimed to investigate the pncA, rpsA and panD gene mutations, the efficiency of the pyrazinamidase (PZAse) enzyme test in determining PZA resistance, the drug susceptibility and their families in PZA-resistant Mycobacterium tuberculosis isolates. Totally 46 PZA resistant M.tuberculosis isolates were included in the study. The pncA, rpsA and panD mutations caused by PZA resistance were investigated by in-house PCR followed by DNA sequencing method. Drug susceptibility was determined with Bactec MGIT 960 (Becton Dickinson, USA) system, the presence of PZAse was evaluated by colorimetric PZAse enzyme assay and the families were determined by the spoligotyping method. Of the 46 PZA-resistant isolates, 24 (52.2%) were identified as PZA monoresistant, 11 (23.9%) multidrug resistant (MDR)-TB and 11 (23.9%) poly drug resistant (PDR)-TB. Gene mutations associated with resistance were detected in 73.9% (34) of PZA-resistant M.tuberculosis isolates. The pncA, rpsA and panD mutations were found in 71.7% (33), 28.2% (12) and 4.3% (2) of the isolates, respectively. The coexistence of pncA/rpsA and pncA/panD gene mutations were determined in 12 and two isolates, respectively. The pncA gene mutations were observed in 3 (33.3%) of 9 (19.6%) isolates whose enzyme presence was detected by the colorimetric PZAse test. In the pncA gene, eight different point mutations in the form of missense mutation;A226C (27.3%), A152C (24.2%), C169G (21.2%) A422C (9.1%), G145A (6.1%), A29G (6.1%), A424G (3%) and T464G (3%) were detected. In the rpsA gene, A636C (42.9%) silent and G1318A (42.9%) missense mutations and in the panD gene, C66G (50%) nonsense and A145G (50%) missense mutations were the most common mutations detected. As a result of genotyping of PZA resistant isolates, the most common genotypes were found in T1 cluster with 17 (36.9%) isolates; followed by the families of Beijing with 7 (15.2%) isolates, H3 with 6 (13%) isolates, TUR with 5 (10.9%) isolates, and LAM 9 with 4 (8.7%) isolates, respectively. In addition, 2 (4.3%) isolates belonging to the ORPHAN family and one isolate belonging to each of LAM TUR, LAM 2, LAM 7, T2, T5-RUS1 families were identified. Our study is the first to investigate all pncA, rpsA and panD gene mutations that have been found to cause PZA resistance in Turkey. Epidemiological studies on PZA resistance will make important contributions to the determination of resistance mechanism and the development of methods that will provide rapid diagnosis for the detection of resistance.