BACKGROUND: The emergence and spread of antimicrobial-resistant (AMR) Neisseria gonorrhoeae (NG) is a major public health concern. In the era of nucleic acid amplifications tests, rapid and accurate molecular approaches are needed to help increase surveillance, guide antimicrobial stewardship, and prevent outbreaks. METHODS: Residual urethral swabs, collected prospectively in the Baltimore City Health Department during a 6-month period, were analyzed by real-time polymerase chain reaction assays for NG DNA and AMR determinants to fluoroquinolones, penicillin, and extended-spectrum cephalosporins. RESULTS: N. gonorrhoeae DNA was detected in 34.8% (73/210) of samples, including 67.3% (68/101) of the swabs that had been previously identified as NG positive by culture. Markers associated with decreased susceptibility to fluoroquinolones were detected in 22.4% of the polymerase chain reaction NG-positive samples. The rate of penicillinase-producing NG was very low (1.6%), and no markers associated with decreased susceptibility to extended-spectrum cephalosporins were detected in this cohort of men using the AMR assays herein described. CONCLUSIONS: Detection of molecular markers associated with AMR in NG can be performed directly from residual clinical samples, although the recovery rate of adequate DNA for molecular testing from these samples can be suboptimal. A high number of samples with mutations associated with decreased susceptibility to fluoroquinolones were identified.