The continued use of pyrazinamide in the treatment of tuberculosis in the absence of a rapid, accurate and standardized pyrazinamide drug susceptibility assays is of great concern. While whole genome sequencing holds promise, it is not yet feasible option in low resource settings as it requires expensive instruments and bioinformatic analysis. We investigated the diagnostic performance of a closed-tube Linear-After-The-Exponential (LATE)-PCR assay for pyrazinamide susceptibility in Mycobacterium tuberculosis. Based on a set of 654 clinical Mycobacterium tuberculosis culture isolates with known mutations throughout the pncA gene as determined by Sanger sequencing, the assay displays excellent sensitivity of 96.9% (95% CI: 95.2–98.6) and specificity of 97.9% (95% CI: 96.1–99.7). In a subset of 384 isolates with phenotypic drug susceptibility testing, we also observed high sensitivity of 98.9% (95% CI: 97.5–100) but lower specificity of 91.8% (95% CI: 87.9–95.8) when compared to phenotypic drug susceptibility testing. We conclude that the LATE PCR assay offers both a rapid and accurate prediction of pyrazinamide susceptibility.