There are no commercially available Food and Drug Administration–cleared rapid tests for Neisseria gonorrhoeae antimicrobial susceptibility testing. This study evaluated the performance of a laboratory-developed real-time polymerase chain reaction assay for genotyping the gyrA gene to determine antimicrobial susceptibility to ciprofloxacin. Validation and clinical performance of the gyrA assay were evaluated across 3 geographic locations (Los Angeles, San Francisco, Philadelphia). Following validation, clinical specimens were collected in Aptima Combo2® CT/NG transport medium from asymptomatic persons who tested positive for Neisseria gonorrhoeae and evaluated for assay percent reportable (i.e., proportion of N. gonorrhoeae–positive specimens that yielded a gyrA genotype). The percentage of gyrA genotyping results differed by laboratory and specimen type. The proportion of specimens that were reportable was best for urine/genital specimens (genotyped = 76.4% (95% confidence interval, 69.9–82%)) followed by rectal (genotyped = 67.2% (95% confidence interval, 63.4–70.6%)) and then pharyngeal specimens (genotyped = 36.1%, (95% confidence interval, 31.9–40.5%)). Overall, asymptomatic patients with N. gonorrhoeae yielded an interpretable genotype 57.2% (784/1370) of the time, of which 480 were wild-type gyrA, resulting in 61% (480/784) being potentially treatable with ciprofloxacin.