Abstract Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is an urgent global health threat. Zoliflodacin is a novel antibiotic undergoing clinical trials for the treatment of gonorrhoea. While there are limited data regarding zoliflodacin resistance in N. gonorrhoeae, three amino acid mutations have been associated with increased MICs of zoliflodacin. Objectives To determine the prevalence of three amino acid mutations associated with zoliflodacin resistance within a large, public database of nearly 13 000 N. gonorrhoeae genomes. Methods PathogenWatch is an online genomic epidemiology platform with a public database of N. gonorrhoeae genomes. That database was used to extract gyrB sequence data and a Basic Local Alignment Search Tool (BLAST) search was performed to identify any of the three amino acid mutations in GyrB that are associated with increased zoliflodacin MICs: D429N, K450N or K450T. As a control for the search methodology, all GyrA sequences were also extracted and S91F mutations were identified and compared with the PathogenWatch database. Results In total, 12 493 N. gonorrhoeae genomes from the PathogenWatch database were included. Among those genomes, none was identified that harboured any of the three mutations associated with increased zoliflodacin MICs. One genome was identified to have a mutation at position 429 in GyrB (D429V). Conclusions The findings suggest that the prevalence of the three mutations associated with zoliflodacin resistance in N. gonorrhoeae is very low. However, further research into the mechanisms of zoliflodacin resistance in N. gonorrhoeae is needed. Genomic epidemiology platforms like PathogenWatch can be used to enhance the global surveillance of AMR.