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Background: Cytomegalovirus (CMV) can vertically transmit from infected mothers to fetuses and causes congenital infection in newborns. Unfortunately, there have been limited data available on the prevalence of congenital CMV (cCMV) infection among Iranian neonates at higher risk of infection. Objectives: The current study aimed to assess the prevalence of cCMV infection among hospitalized neonates in Tehran, Iran, and investigate the diagnostic values of CMV polymerase chain reaction (PCR) on Guthrie cards in comparison to those reported for urine specimens. Methods: This cross-sectional study was carried out on the hospitalized neonates with 3 weeks of age. The urine specimens and Guthrie cards were taken from each eligible newborn. Total nucleic acid was extracted from the samples and tested by PCR for the presence of CMV deoxyribonucleic acid. The cCMV infection was confirmed in the newborns, and the infected neonates underwent further evaluation. Results: Out of 224 newborns, CMV infection was identified in 11 neonates (4.9%). There were no factors in association with cCMV infection. The sensitivity and specificity of dried blood spot (DBS) samples for the identification of newborns with cCMV infection were 90% and 99%, respectively. Conclusions: A significant number of hospitalized neonates in the present study were infected with cCMV infection. The results of the current study revealed that Guthrie cards had adequate sensitivity for the identification of CMV infection in the hospitalized newborns. Since symptomatic newborns with cCMV infection have a higher chance for the development of early- or late-onset sequelae of infection, it is recommended to diagnose and treat this group of newborns.