AIMS To describe the molecular basis of pyrazinamide (PZA) resistance among Mycobacterium tuberculosis in New Zealand, and to compare the pncA gene sequence among isolates with phenotypic (Bactec 960 and Wayne's assay) evidence of PZA resistance. METHODS The pncA gene of 26 clinical isolates of M. tuberculosis found to be PZA resistant by phenotypic methods was sequenced. Phenotypic and genotypic data were compared. RESULTS Mutations were detected in 11 isolates, predominantely among isolates with a multi-drug resistance phenotype. While the mutations were dispersed along the pncA gene, a clustering of mutations were found at amino acid Ser18-His57 and His82-Val128 regions. Seven of the identified mutations have not been described previously. CONCLUSIONS A high diversity of pncA gene mutation was seen among PZA resistant strains of M. tuberculosis. The Bactec 960 phenotypic test may over report PZA resistance.