Mycoplasma genitalium, Mycoplasma hominis, and Ureaplasma urealyticum are associated with infections of the genitourinary tract, reproductive failure. A multiplex PCR was used for detection of these Mycoplasmas species in a single amplification reaction simultaneously. 156 sample were collected from 104 women’s genital and 52 semen specimens (from infertile men) with urogenital infections, referred to gynecological and general outpatient obstetrics clinics. The Specimens were analyzed with species specific primers for identification of M. genitalium, M. hominis, and U. urealyticum by multiplex PCR. The primer sets; amplified a 78 bp fragment (M. genitalium), 280 bp fragment (M. hominis), and 418 bp fragment (U. urealyticumin). Data from the patients were analyzed in order to confirm their relationship of isolates with each disorders. Of the 156 samples examined, the total positive rates of Mycoplasmas were 25% (M. genitalium isolates, 3.8 %; M. hominis isolates, 8.4 %; and U. urealyticum isolates, 12.8 %), by using multiplex PCR. Genital infections caused by mycoplasmas are the most important causes of reproductive disorders. Multiplex PCR was shown to be a highly sensitive, specific test, when specific primer sets were used for screening and diagnosis of genital Mycoplasmas.