Cytomegalovirus is a betaherpesvirus responsible for persistent infections that are generally asymptomatic in healthy individuals. In the absence of an effective immune response, as in neonates, cancer patients, organ transplant recipients, individuals with AIDS, etc., cytomegalovirus may cause severe disease. Early detection of this virus would prevent serious health consequences in immunocompromised patients; it is important to employ sensitive methods and accurate detection to support treatment-related decision making. Real-time molecular methods, such as the polymerase chain reaction, possess higher sensitivity to detect positive samples. We compared the sensitivity and specificity of the following detection methods: the endpoint PCR trade-validated method (Pol, viral gene target) and real-time PCR, which detects viral genes Pol (early gene), and pp65 (late gene). We performed a cross-sectional study of 43 human immunodeficiency virus-positive samples. The molecular detection methods in real-time detected a greater number of cytomegalovirus-positive samples than those at the endpoint. There must be at least two independent cytomegalovirus target-genes in order to make the detection by real-time PCR.