Mycoplasma genitalium (MG) is a sexually transmitted bacterium in which macrolide resistance is rapidly increasing, limiting treatment options. We validated a new assay to detect the presence of macrolide resistance-associated mutations in MG (MG-MRAM). In 2018, symptomatic and asymptomatic clients visiting sexually transmitted infections (STI) clinics in Amsterdam or The Hague were tested for MG using transcription mediated amplification (TMA) assays. The sensitivity to detect MG of the newly developed MG-MRAM qPCR was compared to the MgPa qPCR, both in relation to the TMA assay. For the sensitivity and specificity to detect relevant mutations the MG-MRAM qPCR was compared to 23SrRNA sequencing analysis. The qPCR was subsequently used to determine the presence of MG-MRAM at different anatomical locations and to identify risk factors for MG-MRAM. MG-positive clients (402) providing 493 MG-positive samples were included. In total 309/493 (62.7%) samples from 291 (72.4%) clients were successfully typed with the MG-MRAM qPCR. The MG-MRAM qPCR had a sensitivity of 98.6% (95%CI 91.1%-99.9%) and specificity of 94.1% (95%CI 78.9%-99.0%) to detect MG-MRAM compared to sequencing analysis. Infection with MG-MRAM was detected in 193/291 (66.3%) clients: in 129/178 (72.5%) men and 64/113 (56.6%) women (p = 0.005). Prevalence of MG-MRAM was significantly higher in men, clients with a higher education, HIV-positive clients and clients with >10 sexual partners in the previous six months, but in multivariable analysis no factor was significantly associated with MG-MRAM presence. Since MG-MRAM prevalence was very high, testing for MG-MRAM is essential if treatment for MG is considered, and can be performed with this sensitive and specific qPCR test in routine diagnostics.