Pyrazinamide (PZA) is an important first-line drug used for the short-course treatment of tuberculosis in combination with isoniazid and rifampin. It has been reported that mutations in pncA gene correlate well with PZA resistance depending on the geographic area. On the other hand, different genotypes of Mycobacterium tuberculosis show different affinities to acquire resistance-related mutations. To determine the relative significance of various mutations in the pncA gene in Iranian PZA-resistant M. tuberculosis isolates and to analyze the association of different genotypes of M. tuberculosis with PZA resistance, 34 PZA-resistant M. tuberculosis isolates were analyzed for their pncA mutations using direct sequencing. These isolates were genotyped by IS6110 fingerprinting and spoligotyping methods. Mutations in the pncA gene were identified in 24 of 34 of these isolates (70.58%). No mutations were found in 10 PZA-resistant isolates, which implied that alternative mechanisms of resistance existed in these strains. PZA resistance was strongly (41.2%) associated with multidrug-resistant tuberculosis. Genotyping revealed the Central Asian (CAS) and East-African Indian families as the most prevalent families between PZA-monoresistant isolates versus the Beijing and Haarlem families which were the most frequent families between PZA including multidrug-resistant isolates.