Mycoplasma genitalium (MG) can cause nongonococcal urethritis and is potentially associated with urethritis, endometritis, and cervicitis. Several assays have been developed to detect MG. Molecular amplification assays for organism detection can be problematic due to the potential for false-positive and false-negative results. Confirmatory testing is often required in these situations, requiring additional time and resources. Use of multigene targets could integrate both detection and verification at lower cost. Utilizing two targets, the MgPa adhesion gene and the 16S rRNA gene, a multitarget real-time (MTRT) PCR for the detection of MG was developed. Samples from patients attending sexually transmitted disease clinics were collected in duplicate. Urine samples from males (n = 286) and self-collected vaginal swabs from females (n = 321) were analyzed by MTRT PCR for MG and the Gen-Probe transcription-mediated [corrected] amplification (TMA) assay, which targets MG rRNA for detection (TMA-MG research use only). Utilizing the criteria of any two targets being positively amplified, the MTRT PCR had a sensitivity and specificity of 91.8% (101 positive samples/110 samples tested) and 99.5% (495/497), respectively, with a positive predictive value (PPV) of 98.1% (101/103) and a negative predictive value (NPV) of 98.2% (495/504). The Gen-Probe TMA-MG assay had a sensitivity, specificity, PPV, and NPV of 98.1% (108/110), 98.1% (488/497), 92.3% (108/117), and 99.5% (488/490), respectively. Comparison between the MTRT PCR and TMA-MG assay by kappa statistic analysis indicated that an overall kappa value was 0.941 (95% confidence interval, 0.907 and 0.976). Both assays demonstrated accuracy in the detection of MG from urine samples from male patients and self-collected vaginal swabs from female patients.