Background: Antimicrobial resistance (AMR) of Mycoplasma genitalium (MG) is a growing concern worldwide and surveillance is needed. In Belgium, samples are sent to the National Reference Centre of Sexually Transmitted Infections (NRC-STI) on a voluntary basis and representative or robust national AMR data are lacking. Aim: We aimed to estimate the occurrence of resistant MG in Belgium. Methods: Between July and November 2022, frozen remnants of MG -positive samples from 21 Belgian laboratories were analysed at the NRC-STI. Macrolide and fluoroquinolone resistance -associated mutations (RAMs) were assessed using Sanger sequencing of the 23SrRNA and parC gene. Differences in resistance patterns were correlated with surveillance methodology, sociodemographic and behavioural variables via Fisher's exact test and logistic regression analysis. Results: Of the 244 MG -positive samples received, 232 could be sequenced for macrolide and fluoroquinolone RAMs. Over half of the sequenced samples (55.2%) were resistant to macrolides. All sequenced samples from men who have sex with men (MSM) (24/24) were macrolide-resistant. Fluoroquinolone RAMs were found in 25.9% of the samples and occurrence did not differ between socio-demographic and sexual behaviour characteristics. Conclusion: Although limited in sample size, our data suggest no additional benefit of testing MG retrieved from MSM for macrolide resistance in Belgium, when making treatment decisions. The lower occurrence of macrolide resistance in other population groups, combined with emergence of fluoroquinolone RAMs support macrolide-resistance testing in these groups. Continued surveillance of resistance in MG in different population groups will be crucial to confirm our findings and to guide national testing and treatment strategies. ; Funding The prospective study was financed by the Belgian government. Acknowledgements We would like to thank all laboratory personnel and physicians involved in the study. Moreover, we would like to specifically thank the personnel ...