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Mycoplasma genitalium is an important cause of bacterial sexually transmitted diseases. Diagnosis and susceptibility testing of M. genitalium are limited by the fastidious nature of the organism. Therefore, the prevalence of infection and azithromycin resistance are poorly studied.We conducted an exploratory study on remnant clinical specimens. We collected remnant DNA from consecutive urine samples and clinical swabs (cervical/vaginal, rectal, and pharyngeal) previously tested for Neisseria gonorrhoeae and Chlamydia trachomatis using the Cobas 4800 CT/NG assay (Roche Molecular Systems, Pleasanton, CA) between March-April 2017 from across the University of California, Los Angeles Health System. We then retrospectively tested all specimens with the ResistancePlus MG (550) kit, a molecular assay for the detection of M. genitalium and genetic mutations associated with azithromycin resistance.Among 500 specimens, the prevalence of M. genitalium was 1.1% (95% confidence interval [CI], 0.04%–3.0%) in urine samples (n = 362), 17.4% (95% CI, 5.7%–39.6%) in rectal swabs (n = 23), and 1.9% (95% CI, 0.3%–7.3%) in cervical/vaginal swabs (n = 106). The prevalence of N. gonorrhoeae was 0.6% in urine samples and 4.3% in rectal swabs, whereas the prevalence of C. trachomatis was 2.2% in urine samples, 4.3% in rectal swabs and 3.8% in cervical/vaginal swabs. Of the 10 M. genitalium positive specimens, 8 (80.0%) had a mutation associated with azithromycin resistance.The prevalence of M. genitalium infection in our population varied by anatomic site of infection. Most M. genitalium infections had at least 1 mutation associated with azithromycin resistance.