This study aimed to assess the laboratory diagnosis of Neisseria gonorrhoeae in St. Petersburg, Russia. In total, 334 consecutive symptomatic patients were enrolled. Cervical and urethral specimens from women (n=286) and urethral specimens from men (n=48) were analyzed by microscopy, culture and two in-house NAATs, i.e. polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), developed in Russia. All N. gonorrhoeae-positive samples were confirmed using porA pseudogene and 16S rRNA gene sequencing. All methods displayed 100% specificity, i.e. positive predictive values of 100%. Compared to the PCR (most sensitive method in the present study), in women the sensitivity of both microscopy and culture was 31.8%, and that of NASBA was 90.9%. In men, microscopy, culture and NASBA displayed a sensitivity of 75%, 50% and 100%, respectively. The negative predictive values of microscopy, culture, and NASBA were 97.3%, 97.3%, and 99.6% in women, and 97.8%, 95.7%, and 100% in men, respectively. According to the PCR, the prevalences of N. gonorrhoeae were 4.5% (women) and 8.3% (men). In conclusion, both the investigated Russian NAATs displayed a high sensitivity and specificity. However, in general the diagnosis of gonorrhoea in Russia is suboptimal and crucially requires validation, improvements and quality assurance.