IntroductionGonorrhoea is a major public health concern. With the global emergence and spread of resistance to last-line antibiotic treatment options, gonorrhoea threatens to be untreatable in the future. Therefore, this study performed whole genome characterization ofNeisseria gonorrhoeaecollected in Ghana to identify lineages of circulating strains as well as their phenotypic and genotypic antimicrobial resistance (AMR) profiles.MethodsWhole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) andPUBMLST.org/neisseriadatabases were used to catalogue chromosomal and plasmid genes implicated in AMR. The core genome multi-locus sequence typing (cgMLST) approach was used for comparative genomics analysis.Results and DiscussionIn vitroresistance measured by the E-test method revealed 100%, 91.0% and 85.7% resistance to tetracycline, penicillin and ciprofloxacin, respectively. A total of 22 sequence types (STs) were identified by multilocus sequence typing (MLST), with ST-14422 (n= 10), ST-1927 (n= 8) and ST-11210 (n= 7) being the most prevalent. Six novel STs were also identified (ST-15634, 15636-15639 and 15641). All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A single cefixime-resistant strain, that belongs toN. gonorrhoeaemultiantigen sequence type (NG-MAST) ST1407, a type associated with widespread cephalosporin resistance was identified.Neisseria gonorrhoeaeSequence Typing for Antimicrobial Resistance (NG-STAR), identified 29 unique sequence types, with ST-464 (n= 8) and the novel ST-3366 (n= 8) being the most prevalent. Notably, 20 of the 29 STs were novel, indicative of the unique nature of molecular AMR determinants in the Ghanaian strains. Plasmids were highly prevalent: pTetM and pblaTEM were found in 96% and 92% of isolates, respectively. The TEM-135 allele, which is an amino acid change away from producing a stable extended-spectrum β-lactamase that could result in complete cephalosporin resistance, was identified in 28.5% of the isolates. Using WGS, we characterizedN. gonorrhoeaestrains from Ghana, giving a snapshot of the current state of gonococcal AMR in the country and highlighting the need for constant genomic surveillance.