Cytomegalovirus (CMV) is recognized as the most common congenital viral infection in humans and an important cause of morbidity and mortality in immunocompromised hosts. This recognition of the clinical importance of invasive CMV disease in the setting of immunodeficiency and in children with congenital CMV infection has led to the development of new diagnostic procedures for the rapid identification of CMV. Diagnosis of acute maternal CMV infection by the presence of immunoglobulin (Ig)M and low-avidity IgG requires confirmation of fetal infection, which is typically performed using polymerase chain reaction (PCR) assays for CMV on amniotic fluid. Viral culture of the urine and saliva obtained within the first two weeks of life continues to be the gold standard for diagnosis of congenitally-infected infants. PCR assays of dried blood spots from newborns have been shown to lack sufficient sensitivity for the identification of most neonates with congenital CMV infection for universal screening purposes. However, saliva PCR assays are currently being assessed as a useful screening method for congenital CMV infection. In the immunocompromised host, newer rapid diagnostic assays, such as phosphoprotein 65 antigenemia and CMV real-time PCR of blood or plasma have allowed for preemptive treatment, reducing morbidity and mortality. However, lack of standardized real-time PCR protocols hinders the comparison of data from different centers and the development of uniform guidelines for the management of invasive CMV infections in immunocompromised individuals.