Background: Cytomegalovirus (CMV) is a leading cause of congenital infection in neonates. Most infants with congenital CMV infection are asymptomatic at birth and not diagnosed on routine clinical examination. To identify these at-risk infants early in life, polymerase chain reaction (PCR) assays are done to screen large populations of newborn infants. Objective: We carried out a pilot study to estimate the prevalence of CMV in saliva from newborns by DNA PCR assay. Methods: This study was performed from January 2012 to March 2012 at a maternity hospital in the south of Tehran. All newborns aged between 1 to 14 days born at this hospital were enrolled. Saliva specimens from newborns were collected by swabbing the inside of the baby's mouth and stored at -70°C until PCR processing for virus detection. Results: Six-hundred and twenty infants between 1 to 14 days of age were enrolled during the study period of two months. The PCR assay was positive for CMV in 2 newborns 0.3%. Both of these infants were asymptomatic for congenital CMV at birth and also when followed up at three months and six months of age. Conclusions: Our findings reveal that because of a low yield of positive results, screening for congenital CMV infection would not be cost-effective in Iranian neonates