Mycoplasma genitalium is the cause of an emerging sexually transmitted infection (STI) with high propensity for development of antimicrobial resistance. In a prevalence study conducted at the public STI service in Hong Kong, the first void urine samples of 38 (8%) of 493 male patients with non-gonococcal urethritis (NGU) tested positive for M. genitalium using reverse transcription polymerase chain reaction. Patients with M. genitalium infection were younger [31 vs 33 years, odds ratio (OR) 0.96, 95% confidence interval (CI) 0.93-0.996; P=0.03], more likely to present with urethral discharge (12% vs 6%, OR 2.16, 95% CI 1.10-4.23; P=0.02) and had symptom duration >2 weeks (14% vs 6%, OR 2.34, 95% CI 1.10-4.97; P=0.03) compared with patients without M. genitalium infection. The prevalence of M. genitalium infection was lower in patients co-infected with Chlamydia trachomatis compared with patients with isolated infection (4% vs 10%, OR 0.38, 95% CI 0.17-0.84; P=0.02). The prevalence of M. genitalium infection was not higher in men who have sex with men. Antimicrobial-resistance-conferring mutations were present in 24 (63%) patients with M. genitalium - 23S rRNA 18 (47%) and parC 19 (53%). Similar to neighbouring countries in the Asia Pacific region, concurrent resistance mutations against both macrolides and fluoroquinolones were demonstrated in 14 (37%) patients. Histories of azithromycin and moxifloxacin use were significantly associated with a diagnosis of M. genitalium infection. Characteristically, NGU in Hong Kong featured the co-existence of mono-resistance against macrolides or fluoroquinolones, and the presence of dual class resistance. The geographic variability of antimicrobial resistance against M. genitalium is attributed not just to the different transmission networks formed in separate population groups, but the antimicrobial prescriptions for the treatment of urethritis in the community.