B A C K G ROU N D : Multidrug-resistant tuberculosis (MDR-TB) regimens often contain pyrazinamide (PZA) even if susceptibility to the drug has not been confirmed. This gap is due to the limited availability and reliability of PZA susceptibility testing. OB J E C T IVE S : To estimate the prevalence of PZA resistance using the Wayne assay among TB patients in Lima, Peru, to describe characteristics associated with PZA resistance and to compare the performance of Wayne with that of BACTECe MGITe 960. METHODS : PZA susceptibility using the Wayne assay was tested in patients diagnosed with culture-positive pulmonary TB from September 2009 to August 2012. Factors associated with PZA resistance were evaluated. We compared the performance of the Wayne assay to that of MGIT 960 in a convenience sample. RESULT S : The prevalence of PZA resistance was 6.6% (95%CI 5.8-7.5) among 3277 patients, and 47.7% (95%CI 42.7-52.6) among a subset of 405 MDR-TB patients. In multivariable analysis, MDR-TB (OR 86.0, 95%CI 54.0-136.9) and Latin American-Mediterranean lineage (OR 3.40, 95%CI 2.33-4.96) were associated with PZA resistance. TheWayne assay was in agreement with MGIT 960 in 83.9% of samples (j 0.66, 95%CI 0.56-0.76). CONCLUS ION: PZA resistance was detected using the Wayne assay in nearly half of MDR-TB patients in Lima. This test can inform the selection and composition of regimens, especially those dependent on additional resistance.