Human cytomegalovirus (CMV) is the most frequent cause of congenital infection in humans. In the first prevalence study of congenital CMV infection in Eastern and Central Europe, all neonates born in a 22-month period in two Slovenian maternity units (total of 2,841 newborns) were screened prospectively for congenital CMV infection by a real-time polymerase chain reaction (PCR) in urine. In all newborns with positive screening results, plasma and dried blood spots (DBS) collected at birth were tested additionally for CMV DNA. Congenital CMV infection was confirmed by virus isolation from a urine sample collected within the first 2 weeks of life. Congenital CMV infection was identified in four out of 2,841 newborns tested (incidence 0.14%; 95% CI, 0.05-0.39%). In four newborns with confirmed congenital infection, the concentration of CMV DNA in urine ranged from 4.68 to 8.18 log(10)  copies/ml, all four newborns had detectable CMV DNA in plasma taken at birth (1.26-3.34 log(10)  copies/ml) and two out of four had detectable CMV DNA in DBS collected during newborn metabolic screening. None of the four newborns with confirmed congenital CMV infection was symptomatic. The study showed that the prevalence of congenital CMV infection at birth in Slovenia is among the lowest in the world and that CMV DNA PCR testing of urine is a suitable and affordable real-time screening strategy for congenital CMV infection. If it is performed in 24 mini-pools, the cost of screening is 1.4 €/newborn and the cost of detecting a single newborn with congenital CMV infection 1,000 €.