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This before-and-after study measured the impact of a change in testing methods from culture to nucleic acid amplification testing (NAAT) on the detection of pharyngeal and rectal gonorrhea in men who have sex with men (MSM) on a sexual health service level, including the effect on subgroups anticipated to have higher rates of gonorrhea.In March 2015, Melbourne Sexual Health Centre changed its laboratory method for gonococcal testing from culture to NAAT using the Aptima Combo 2 and Aptima GC tests. We compared the proportion of tests positive for rectal and pharyngeal gonorrhea in MSM using culture in 2014 with those using NAAT in 2015.The proportion of tests positive for rectal gonorrhea by NAAT was double that obtained by culture (8% vs 3.9%; prevalence ratio [PR], 2.0; 95% confidence interval [CI], 1.8–2.4) and 5-fold for pharyngeal gonorrhea (8.3% vs 1.6%; PR, 5.2; 95% CI, 4.2–6.4). Similar increases in test positivity were observed in human immunodeficiency virus (HIV)-positive and HIV-negative men. By NAAT, test positivity for rectal gonorrhea was higher in HIV-positive compared with HIV-negative men (15.4% vs 7.3%; PR, 2.1; 95% CI, 1.7–2.6). Culture and NAAT had similar test positivity for rectal gonorrhea among men who reported contact with gonorrhea (24.9% vs 25.3%, PR 1.0, 95% CI 0.8–1.4) and men who presented with symptoms of proctitis (22.2% vs 27.9%, PR 1.3, 95% CI 0.8–2.0).A switch from culture to Aptima Combo 2 testing for extragenital gonorrhea in MSM increased detection and was most marked for pharyngeal infections.