Human cytomegalovirus (CMV) is a clinically important pathogen that causes significant morbidity and mortality in immunocompromised patients and is typically monitored using real-time polymerase chain reaction (real-time PCR). International standards and certified reference materials were recently developed by the WHO, providing the opportunity to standardize viral load reporting. Clinical microbiologists who conduct quantitative CMV DNA testing should be aware of technical issues that can affect the analytical and clinical performance of the method used. These include specimen type, limits of detection and quantification, linear range, reproducibility, and wide variability in viral load values among different assays. Specimen types for testing include whole blood, plasma, serum, and peripheral blood leukocytes. The tests that use whole blood and peripheral blood leukocytes have higher sensitivities. Adsorption chromatography methods are widely used for nucleic acid extraction, and efficiencies can differ between manual and automatic processes. The most common method for quantitative CMV DNA testing is real-time PCR. All CMV testing methods require quality control at the pre-analytic, analytic, and post-analytic stages. In transplant patients, specific quantitative results and monitoring of any changes at follow-up are important. Five to seven days is an adequate follow-up interval in this regard, and drug-resistant CMV should be suspected if there is no response to therapy. One specimen type should be chosen for follow-up quantitative CMV DNA testing, optimized according to WHO standards. Further studies are needed to better standardize CMV testing approaches and to determine the appropriate clinical cut-off level. (Ann Clin Microbiol 2017;20:21-26)