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The oropharynx represents a crucial site for the emergence of multi-drug resistance in Neisseria gonorrhoeae. The mosaic penA alleles, associated with decreased susceptibility to cephalosporins, have emerged by DNA recombination with partial penA genes, particularly those from commensal pharyngeal Neisseria species. Here, we investigated the prevalence of the mosaic structure of the penA gene in the oropharynx of men who have sex with men testing negative for pharyngeal gonorrhoea. From January 2016 to June 2018, 351 gonorrhoea-negative men who have sex with men attending a sexually transmitted infection clinic in Italy were enrolled. Pharyngeal swabs underwent a real-time polymerase chain reaction (PCR) for the detection of the mosaic penA gene. In case of positivity, PCR products were sequenced and searched against several sequences of Neisseria strains. Overall, 31 patients (8.8%) were found positive for the presence of the mosaic penA gene. The positivity was significantly associated with previous cases of pharyngeal gonorrhoea (relative risk [RR]: 3.56, 95% confidence interval 1.44–8.80) and with recent exposure to beta-lactams (RR: 4.29, 95% confidence interval 2.20–8.38). All penA-positive samples showed a high relatedness (90–99%) with mosaic-positive Neisseria strains. Our data underline that commensal Neisseria species of the oropharynx may be a significant reservoir for genetic material conferring antimicrobial resistance in N. gonorrhoeae.