Background and purpose: Ureaplasma urealyticum is one of the most common causes of Non-Gonococcal Urethritis (NGU). Asymptomatic clinical infection caused by this bacterium can cause malnutrition of the sexual attachment glands and its presence in semen contributes to lower fertility. The aim of this study was to identify Ureaplasma urealyticum in semen of infertile men using PCR method as an accurate diagnostic method. Materials and methods: The PCR test was optimized by standard strain to detect Ureaplasma urealyticum and then was studied in terms of specificity and limit of detection (LOD). Semen samples were collected from 100 infertile men and each sample was divided into two parts: the first part was tested by semen analysis and the second part was tested by PCR method. DNA was extracted using phenol-chloroform method and the PCR test was done for detection of Ureaplasma urealyticum. Results: Among the semen samples, 16 cases (16%) were found to be positive for Ureaplasma urealyticum. According to the spermogram test, the leukocyte level was also more than normal level in these samples (0-1 Mil/ml). Conclusion: Screening of infertile couples for Ureaplasma urealyticum infection in those without clinical symptoms, thereby performing timely antibiotic therapy play key roles in treatment of infertility. Hence, PCR method is introduced as a valuable and reliable technique to identify Ureaplasma urealyticum.