The data presented in this article demonstrate how the polymerase chain reaction (PCR) can be used to detect human cytomegalovirus (HCMV) DNA in the plasma and cerebrospinal fluid (CSF) of infected individuals. Detection of HCMV DNA in plasma of transplant recipients and persons infected with human immunodeficiency virus identifies people with acute visceral disease and those at highest risk for development of HCMV disease. The detection of HCMV DNA in CSF is of particular help in identifying persons with HCMV-related central nervous system disease. In addition, specific mutations within the UL97 region, which encodes for a HCMV protein kinase, have been found to confer ganciclovir resistance and can be detected in plasma and CSF by direct sequencing of PCR products. These data will help provide tools for clinicians to better diagnose, manage, and treat persons with HCMV disease.