Cross-reactions of gonococcal nucleic acid amplification tests (NAATs) with commensal Neisseria strains are well documented. Recent data now indicate that sequence-related false-negative results can occur in gonococcal NAATs, whereby target sequences either are absent or contain several mutations. In this study, a duplex Neisseria gonorrhoeae real-time polymerase chain reaction (PCR) (NGduplex) assay targeting the gonococcal porA pseudogene and multicopy opa genes was developed. The NGduplex was evaluated by testing 596 clinical specimens, including 292 urogenital specimens and 304 throat swab specimens. The results were compared with those obtained using a consensus reference standard comprising 3 monoplex real-time PCR assays. The results show that the NGduplex assay is highly suitable for routine screening for gonorrhea, providing an overall clinical sensitivity and specificity of 100% and 99.3%, respectively, for both urogenital and throat swab specimens. In addition, the 2-target system of the NGduplex assay decreases the potential for sequence-related false-negative results and can provide simultaneous confirmation of positive results.