Abstract: Ureaplasma urealyticum is a causative agent of non-gonococcal urethritis, prostatitis, epididymitis and infertility. The organism is more common in partners of infertile than fertile marriages. U. urealyticum infections not only jeopardize fertility but also pose a risk for infertility treatment and resulting pregnancies. The purpose of this study was to determine the prevalence of U. urealyticum in semen of infertile and healthy men by Polymerase Chain Reaction (PCR). Semen samples were obtained from infertile patients and healthy control and were subjected to the routine andrological analysis and PCR. DNA was extracted by Cadieux method and analyzed by PCR protocol with species-specific primers for U. urealyticum (urease gene). U. urealyticum was detected significantly by PCR in 12 of 100 (12%) semen specimens from infertile patients and in 3 of 100 (3%) healthy men. The volume of semen fluid, concentration of sperm cells and sperm cell with normal morphology were significantly decreased in infertile men. In the group of infertile patients with PCR positive for U. urealyticum the volume, count and morphology of semen samples were lower than in the infertile patients with PCR negative results.