ABSTRACTTo explore the phenotypic and genotypic characterization of pyrazinamide (PZA) resistance among multidrug-resistantMycobacterium tuberculosis(MDR-TB) isolates in Zhejiang province, a total of 274 MDR-TB isolates were collected. Drug susceptibility testing and spoligotyping were performed on all clinical isolates. In addition, the mutated features of PZA-resistant loci, includingpncAandrpsA, were also analyzed by DNA sequencing. Our results showed that the prevalence of PZA resistance among MDR-TB strains in Zhejiang province was 43.07% and that PZA resistance was associated with concomitant resistance to streptomycin. The majority of PZA-resistant MDR-TB isolates belonged to the Beijing family. Mutations withinpncA, notrpsA, constituted the primary mechanism of PZA resistance. Among 118 PZA-resistant isolates, 53 different mutations were observed inpncA, and most of them were point mutations. Compared with the phenotypic data, DNA sequencing ofpncAhas sensitivity and specificity of 77.97% and 96.79%, respectively. Analysis ofpncAprovided a robust tool for rapid detection of PZA drug resistance.