Following analysis of primary cervix, vagina, and first-void female urine specimens for Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis via commercial transcription-mediated amplification (TMA), residual material was subjected to Mycoplasma genitalium research-use-only TMA. Representation within a 2,478-specimen retrospective study set was established by comparison to a 6-month audit of clinical C. trachomatis TMA (12,999 specimens) on the basis of the C. trachomatis detection rate, specimen source distribution, clinic location, and age. M. genitalium was detected in 282 (11.4%) patients. This rate was higher than those seen with T. vaginalis (9.0%; P ‫؍‬ 0.005), C. trachomatis (6.2%), and N. gonorrhoeae (1.4%). Positive M. genitalium results were confirmed by repeat testing or alternative-target TMA at a rate of 98.7%. The mean age of the M. genitalium-infected females (24.7 years) was lower than that of the T. vaginalis-infected females (mean, 30.1 years; P < 0.0001) and higher than that of the C. trachomatis-infected females (mean, 23.8 years; P ‫؍‬ 0.003). Of 566 patient encounters positive for at least one sexually transmitted infection (STI), 35.9% exhibited sole detection of M. genitalium (P < 0.0004 versus sole detection of other STI agents) and 26.1% were solely positive for T. vaginalis (P < 0.0002 versus C. trachomatis). The M. genitalium and T. vaginalis detection rates among 755 patients at urban emergency departments were 14.6% and 13.0%, respectively (P ‫؍‬ 0.37). A 10.0% M. genitalium detection rate from other facilities exceeded that of T. vaginalis (7.2%; P ‫؍‬ 0.004). Incorporation of M. genitalium TMA into comprehensive testing programs would detect M. genitalium in a significant proportion of females, particularly those in outpatient obstetrics and gynecology (OB/GYN) settings. T he sexually transmitted infection (STI) agentMycoplasma genitalium has historically had a role of pathogenicity in male nongonococcal urethritis (1). Recent evidence has implicated the bacterium in clinically significant disease in females (2, 3). Additional studies suggest that M. genitalium infection promotes HIV acquisition (4-6) and virus shedding (7,8). Moreover, in a recent meta-analysis, Lis et al. (9) reported significant associations between M. genitalium infection and cervicitis, pelvic inflammatory disease, preterm birth, and spontaneous abortion.Until recently, a lack of reliable testing options has curtailed laboratory diagnosis of M. genitalium infection. Culture and serologic modalities have been limited by sensitivity and/or cross-reactivity with other mycoplasmas (1) and are becoming supplanted by molecular diagnostics, largely on a research basis. PCR-based assays have correlated M. genitalium DNA burden with clinical condition (10, 11) and treatment efficacy (12) in males. Quantitative molecular analysis has sought to study progression of genital disease in females (13). In the realm of laboratory diagnosis, initial studies of target capture-based transcription-mediated amp...