Due to the poor positive predictive value of nucleic acid amplification tests (NAATs) for gonorrhoea when applied to a low-prevalence setting, current guidelines recommend the use of supplementary polymerase chain reaction (PCR) targeting a different gene for confirmation of true positives in urogenital specimens. This study sought to standardize and evaluate performance of an in-house opa gene-based PCR assay for gonorrhoea compared to assays targeting the porA pseudogene and 16S rRNA gene. Four hundred samples (300 endocervical, 100 urethral swabs) from patients attending STD clinics in New Delhi, India were used. The sensitivity, specificity, positive predictive value and negative predictive value of the opa-based PCR were 100%, 97·9%, 89·5% and 100%, respectively. In females, the use of NAATs provided enhanced diagnosis of gonorrhoea.