A complex method for detection of genetic markers of N. gonorrhoeae resistance to penicillin was developed. Mutations in penA and ponA genes were detected by minisequencing reaction with subsequent detection of reaction products by MALDI-TOF mass spectrometry. This approach was tested on 31 clinical strains of N. gonorrhoeae with minimum inhibitory concentration of penicillin from 0.03 to 8 μg/ml and higher. Mutations in penA and ponA genes in moderately resistant strains were shown (minimum inhibitory concentration up to 0.5 μg/ml) and mutations in penA, ponA, and penB genes in resistant strains (minimum inhibitory concentration more than 1.0 μg/ml). β-Lactamase genes were detected in 4 strains with high resistance (minimum inhibitory concentration 4–8 and more μg/ml). Correlation between microbiological resistance and presence of respective mutations in the studied locuses was detected.