Ventilator-acquired pneumonia (VAP) remains a challenge to intensive care units, with secure diagnosis relying on microbiological cultures that take up to 72 hours to result. We sought to derive and validate a novel, real-time 16S rRNA gene polymerase chain reaction (PCR) for rapid exclusion of VAP. Bronchoalveolar lavage (BAL) was obtained from two independent cohorts of patients with suspected VAP. Patients were recruited in a two-centre derivation cohort and a 12-centre confirmation cohort. Confirmed VAP was defined as growth of >104 colony forming units/ml on semi-quantitative culture and compared to a 16S PCR assay. Samples were tested from 67 patients in the derivation cohort, 10 (15%) of whom had confirmed VAP. Using cycles to cross threshold (Ct) values as the result of the 16S PCR test, the area under ROC curve (AUROC) was 0.94 (95% CI 0.86-1.0 p<0.0001). Samples from 92 patients were available from the confirmation cohort, 26 (28%) of whom had confirmed VAP. The AUROC for Ct in this cohort was 0.89 (95% CI 0.83-0.95 p<0.0001). This study has derived and assessed the diagnostic accuracy of a novel application for 16S PCR. This suggests that 16S PCR in BAL could be used as a rapid test in suspected VAP and may allow better stewardship of antibiotics.