Basis of pyrazinamide (PZA) resistance in M. tuberculosis is commonly associated with mutations in the pncA gene encoding pyrazinamidase (PZase) enzyme. The complete pncA open reading frame of 0,6 kb from R2 and R6 clinical isolate of PZA-resistant Mycobacterium tuberculosis has been characterized. A group mutations of T40C, G419A and A535G which link to amino acid replacements of Cis14Arg, Arg140His, Ser179Gly was identifed in pncA gene from the R2 PZA-resistant Mycobacterium tuberculosis, meanwhile a G511A mutation which replace amino acid of Ala171Thr was found in pncA gene from the R6 PZA-resistant strain. The structure modelling study to determine the effect of amino acid substitutions in the R2 and R6 PZase was carried out. Superposition of the generated model for R2 and R6 PZase with the wild-type PZase structure gave root mean square deviations (RMSD) of 0.24 and 0.23°A respectively, suggesting highly similar structures. However, the mutant structures of R2 and R6 PZase amended several interactions in its wild-type structure, expecially in the regions that contain the catalytic residues. The physical-chemical changes around the active site may be unfavorable for R2 and R6 PZase activities, and in turn create PZA resistance in both M. bacterium strains.