BACKGROUND: Neisseria gonorrhoeae, the aetiological agent of gonorrhoea, is an increasing global health priority due to high levels of antimicrobial resistance (AMR). It is estimated that up to 42% of patients are infected at multiple anatomical sites simultaneously. Previous studies identified that 7%-40% of those with multisite infection have different strains infecting different sites, with potentially different antimicrobial susceptibility profiles. This study aims to estimate the proportion of patients with multisite infection through differential antimicrobial susceptibility testing (AST) profiles and sequence-based molecular methods. METHODS: This was a cross-sectional study of multisite gonococcal isolates provided by three National Health Service laboratories. Minimum inhibitory concentrations (MICs) for cefixime, ceftriaxone, azithromycin, ciprofloxacin, tetracycline and spectinomycin were determined. Possible multistrain infections were defined as isolates with a significant difference in MIC to at least one antimicrobial. Whole genome sequencing (WGS) was performed to determine multistrain infection through N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR), multilocus sequencing typing (MLST) and single nucleotide polymorphism (SNP) phylogeny, and to compare AST profiles with identified AMR genes. RESULTS: Ninety-one isolates were collected from 41 patients with multisite infections. Of these 41 patients, 6 (14.6%) had N. gonorrhoeae isolates with discordant MICs. WGS-based typing confirmed that four out of six patients were infected with different gonococcal strains. The relatedness of isolates with the same MLST across multiple patients was differentiated using SNP-based analysis, and this included the identification of a potential transmission event. WGS-based AMR prediction for all antimicrobials tested correlated well with the phenotypic data. CONCLUSION: This study demonstrates that potentially a significant proportion of patients with multisite infections are infected with multiple gonococcal strains, with differing AST profiles, at different anatomical sites. This has implications for patient sampling, susceptibility testing protocols, AMR surveillance and potentially appropriate antibiotic therapy.