Abstract We developed a PCR-based assay involving Invader® technology for detection of the genital mycoplasmas of Mycoplasma genitalium , Mycoplasma hominis , Ureaplasma urealyticum , and Ureaplasma parvum . We compared its performance with that of a PCR-microtiter plate hybridization assay, which we developed previously, in detecting genital mycoplasmas in first-voided urine (FVU) specimens from men with non-gonococcal urethritis. The tests targeting each of the genital mycoplasmas were specific for the respective species and could detect as few as 10 copies of the plasmids containing the target genes of each of the genital mycoplasmas per reaction. The assay using the InvaderPlus® method (InvaderPlus® assay) showed very similar performance to that of the PCR-microtiter plate hybridization assay for detecting the genital mycoplasmas in the FVU specimens. In addition, the PCR and endonuclease reaction in the InvaderPlus® assay were carried out simultaneously in one procedure, thus simplifying the assay, leading to time- and labor-savings and a decrease in the risk of specimen contamination. The InvaderPlus® assay could be useful in diagnosing genitourinary tract infections caused by the genital mycoplasmas.