Objectives Reference laboratories are increasingly using more sensitive rapid molecular techniques, such as nucleic acid amplification tests (NAATs), to diagnose infections with Neisseria gonorrhoeae. We determined the proportion of patients at sentinel genitourinary medicine clinics in England whose NAAT-positive diagnoses were also culture-positive for N. gonorrhoeae, and investigated whether they differed from those that were not. Methods Behavioural and clinical data from all NAATpositive patients reported from 23 clinics included in the Gonoccocal Resistance to Antimicrobials Surveillance Programme from July to September 2012 were included in this analysis. Unadjusted and adjusted associations between patient characteristics and culture-positive infection with N. gonorrhoeae were determined. Results Of 3076 NAAT-positive patients, 46.4% had culture-positive infections. Most NAAT-positive patients were 35 years old (73.0%), white (67.9%), and men who had sex with men (60.1%). Women and men who had sex with men were less likely than heterosexual men to have culture-positive infections (adjusted OR (95% CI) 0.53 (0.41 to 0.68), p0.001; and 0.74 (0.59 to 0.93), p=0.010, respectively), while those who were symptomatic (4.61 (3.92 to 5.42), p0.001), and those presenting with infection at multiple sites (2.15 (1.76 to 2.62), p0.001) were more likely to have culture-positive infections. Conclusions Although gonococcal isolates were available from almost half of the NAAT-positive patients, culture was not attempted or may have failed in the remainder. Patients with culture-positive isolates were not representative of all NAAT-positive patients. Routine culture is necessary for monitoring emerging antimicrobial resistance and to inform gonorrhoea treatment guidelines.