Mycoplasmas hominis, Mycoplasmas genitalium and Ureaplasma urealyticum are associated with infections of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. A Multiplex PCR was developed for simultaneously detection of theseMycoplasmas species in a single amplification reaction. The total number of 104 samples was collected from 104 women’s genital specimens with urogenital infections for identification of M.hominis, M.genitalium and U.realyticum by Multiplex PCR. The High Pure PCR Template Preparation Kit purified nucleic acids from 100 ml of specimen (American, Roche Company).In addition to the kit, boiling method was used to extraction of DNA from samples.UUA2 and UUS2 primers were used for urease gene amplification of U.urealyticum, MH1 and MH2 used for 16S rRNA gene amplification of M.hominis, and Adhesion protein gene (MgPa) used for 16S rRNA gene amplification of M.genitalium in all samples. The number of 28 samples (27%) was positive for Mycoplasmas. M.hominis, M.genitalium, and U.urealyticum were detected in 8.7, 3.9, and 14.5 percent of samples, respectively. The accumulated frequencies for M.hominis, M.genitalium, and U.urealyticum were 9 (8.7%), 4 (3.9%), and 15 (14.5 %), respectively. The results of this study revealed that Multiplex PCR is a highly sensitive, specific and cost-effective test for screening of genitourinary tract infections.