Cytomegalovirus (CMV) is the most common congenital infection in humans and an important cause of morbidity and mortality in immunocompromised hosts. Congenital CMV infection occurs in approximately 0.5 to 1 percent of all newborns in the United States and can result in significant neurological sequelae. The gold standard for diagnosing congenital CMV infection is isolation of the virus from infants within the first 2 weeks of life through conventional or rapid cell culture techniques. Newer molecular diagnostic methods to diagnose congenital CMV infection, including the nucleic acid amplification of viral DNA from the peripheral blood of infants, are being investigated, and the preliminary results show promise. However, more work must be done to standardize and validate these methods before they can be used routinely in establishing the diagnosis of congenital CMV infection.