Aim: Genital mycoplasmas are known as sexually transmitted agents, causing mainly urethritis, pelvic inflammatory disease, spontaneous abortion, pyelonephritis, infertility, still birth, low birth weight, neonatal meningititis, and neonatal pneumonia. Mycoplasma infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. Diagnosis of genital mycoplasma infections by bacterial conventional methods is very difficult. The aim of this study was to comparison of culture with polymerase chain reaction (PCR) for to determine the prevalence of Ureaplasma urealyticum and Mycoplasma hominis in the endocervical specimens from infertile women. Methods: 312 endocervical swab samples were taken from infertile women, and transported with a mycoplasma transport media. The culture was done with liquid-solid method. DNA was extracted by Cadieux method, and analyzed by PCR protocol with species-specific primers. Results: Genital mycoplasmas were detected in 35.5%( 111/312) specimens by both culture and PCR methods.16%(50/312) samples were PCR positive as well as culture positive, 23%(72/312) were positive only by PCR, and 2.8%(9/312) were positive only by culture. The sensitivity of 91.8% and 53% were found for PCR and culture respectively. Of the 111 positive specimens, 59 (53%) were positive only for U. urealyticum, 29(26%) were positive only for M. hominis and 23(20.7%) presented both organisms. Conclusion: Because of the potential adverse effects of mycoplasmas on the success rate of highly specialized infertility treatment, and their causal roles in several maternal complications of pregnancy and in neonatal morbidity and mortality, the rapid detection of mycoplasmas by PCR in infertile women could be important and necessary. The increased sensitivity and shorter time requirement of PCR support its further development for the diagnosis of mycoplasmas infections.