Abstract: Mycoplasma genitalium (MG) infections are a growing concern within the field of sexually transmitted infections. However, diagnostic assays for MG have been limited in the United States (US). As most infections are asymptomatic, individuals can unknowingly pass the infection on and the prevalence is likely to be underestimated. Diagnosis of MG infection is recommended using a nucleic acid test. This multicenter study assessed the performance of the cobas® TV/MG assay (cobas) for the detection of MG, using 22,150 urogenital specimens from both symptomatic and asymptomatic men and women collected at geographically diverse sites across the US. The performance was compared to a reference standard of three laboratory-developed tests (LDTs). The specificity of the cobas assay for MG ranged from 96.0% to 99.8% across symptomatic and asymptomatic men and women. The sensitivity in female vaginal swabs and urine samples was 96.6% (95% confidence interval [CI] 88.5–99.1%) and 86.4% (95% CI 75.5–93.0%), respectively. The sensitivity in male urine and meatal swab samples was 100% (95% CI 94.0–100%) and 85.0% (95% CI 73.9–91.9%), respectively. This study demonstrated that the cobas assay was highly sensitive and specific in all relevant clinical samples for the detection of MG.