OBJECTIVES: We aimed to estimate the prevalence of infection and of mutations linked to macrolide resistance using the ResistancePlus MG assay (SpeeDx, Sydney, New South Wales, Australia) in first-void urine (FVU), anorectal and oropharyngeal samples from men who have sex with men (MSM) attending Western Sydney Sexual Health Centre (WSSHC). METHODS: Consecutive symptomatic and asymptomatic MSM attending for STI testing were prospectively enrolled. testing using the ResistancePlus MG assay was performed on FVU, anorectal and oropharyngeal samples routinely collected for and assays. RESULTS: Overall, the prevalence of infection in the study group was 13.4% (68/508). Most (79.4%, 54/68) harboured macrolide resistance mutations (87.5% of urethral and 75.6% of anorectal infections). The anorectum was the most commonly infected site (45/505, 8.9%), followed by the urethra (24/508, 4.7%). No oropharyngeal infections were detected (0/508). Most of the anorectal (93.3%) and urethral (79.2%) infections were asymptomatic.MSM who were taking HIV pre-exposure prophylaxis (PrEP) were twice as likely to be infected with compared with MSM who were not on PrEP (OR 2.1, 95% CI 1.3 to 3.6; P=0.0041). Always using condoms for anal sex in the last 3 months was protective of infection (OR 0.8, 95% CI 0.6 to 1.0; P=0.0186). CONCLUSIONS: We demonstrated a high prevalence of and very high levels of macrolide resistance among MSM attending WSSHC. Our findings support the routine use of an assay to detect macrolide resistance mutations in infections. This will ensure, in regions or populations with high rates of macrolide resistance among strains, that first-line treatment with azithromycin will only be used if a macrolide-sensitive strain is identified.