This prospective, multicenter clinical trial was conducted to compare the performance of the cobas® 4800 CT/NG, APTIMA Combo 2®, and ProbeTec™ ET CT/GC assays for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in Japan. From 1274 male and female patients, more than 1900 urine, endocervical and throat specimens were collected. Positive and negative concordance rates for CT and NG results obtained for urine and endocervical samples collected from the same patient were high in all three assays (range 96.0–99.6%). The accuracy of the cobas® 4800 CT/NG test did not differ significantly from that of the APTIMA Combo 2® and ProbeTec™ ET CT/GC assays. The accuracy of the assays did not change depending on the order of collection of endocervical specimens. Concordance rates for results obtained for throat swabs and mouthwashes in the ProbeTec™ ET CT/GC and cobas® 4800 CT/NG assays, respectively, were 98.8% for CT and 95.1% for NG. These data suggest that the cobas®4800 CT/NG test is a reliable and highly accurate diagnostic tool for the detection of CT and NG in urine, genital, and oral specimens. Owing to the high correlation of urine and endocervical swab results and the ease of acquisition, urine samples are suggested as the specimen of choice for screening of CT and NG.