Background: Gonorrhoea is one of the most prevalent sexually transmitted infection (STI) across the world. Isolation and preservation of gonococci is important to perform antimicrobial susceptibility testing and molecular characterization. Objective: To evaluate the preservation of gonococci by deep freezing, chocolate GC agar slope and lyophilization. Methods: The study was undertaken at State level STI/RTI Laboratoryat a tertiary care hospital. Isolation of gonococci was done from suspected cases of gonorrhoea. The culture isolates of gonococci were preserved by deep freezing, chocolate GC agar slope and lyophilization. Results: Out of 71 suspected cases of gonorrhoea, N. gonorrhoeae was isolated from 22 cases (30.9%). Preservation of all 22 isolates of N. gonorrhoeae were attempted by 3 methods.Out of 22 gonococcal isolates preserved by deep freezing (-700 C), 12 (54.5%), 06 (27.2%) and 03 (13.6%) isolates were revived at 3 months, 6 months and 8 months respectively by nutrient broth - glycerol method; 10 (45.5%), 04 (18.1%) and 01 (4.5%) were revived at 3 months, 6 months and 8 months respectively by preservation media (trypticase soy broth and yeast extract). Out of 22 gonococcal isolates, 18 (81.8%), 14 (63.63%) and 06 (27.27%) isolates were revived at end of 1 month, 3 months and 6 months by subculturing gonococcal isolates from chocolate GC agar slope. Conclusion: Chocolate GC agar slope is preferred method for preservation of gonococcal isolates over other methods in routine microbiology laboratories as its inexpensive, less laborious, doesn’t require sophisticated equipment and recovery rate of gonococcal isolates is good upto 6 months.