© Lippincott Williams & Wilkins. Background Nucleic acid amplification testing (NAAT) is the preferred method to detect Chlamydia trachomatis and Neisseria gonorrhoeae, but information regarding performance of currently available assays is needed. This study evaluated the performance of the Aptima Combo 2, GeneXpert, cobas4800, and ProbeTec QX (CTQ/GCQ) to detect chlamydia and gonorrhea in pharyngeal, rectal, and urine specimen. Methods Adult male patients seen at an urban human immunodeficiency virus clinic in Birmingham, Alabama who reported sex with men (men who have sex with men) and no antibiotic use in the past 30 days were enrolled between November 2014 and December 2016. Following a baseline survey, rectal and initial void urine specimens were self-collected. A composite infection standard was used, where 1 assay was compared with 3 others to determine sensitivity and specificity estimates for rectal and urine samples. Two pharyngeal samples were clinician-collected for chlamydia and gonorrhea testing and both had to be positive to be considered a true positive. Results Among the 181 men enrolled into the study, 15.5% and 7.2% had at least 1 positive chlamydia and gonorrhea result at any site, respectively. Among all 4 assays, chlamydia sensitivity rates ranged from 82% to 96% among rectal samples. Rectal gonorrhea sensitivity estimates ranged from 67% to 99%. The GCQ assay was less sensitive in detecting rectal gonorrhea compared with the other assays (P = 0.02). Conclusions More than 80% of chlamydia and gonorrhea infections would have been missed with urine-only screening, highlighting the importance in using NAATs to detect chlamydia and gonorrhea infections among men who have sex with men.