Human Cytomegalovirus (HCMV) infection is life-threatening for immunocompromised patients. Quantitative molecular assays on whole blood or plasma are the gold standard for the diagnosis of invasive HCMV infection and for monitoring antiviral treatment in individuals at risk of HCMV disease. For these reasons, an accurate standardization toward the WHO 1st International Standard among different centers and diagnostic kits represents an effort for better clinical management of HCMV-positive patients. Herein, we evaluate, for the first time, the performance of a new transcription-mediated amplification (TMA) assay versus quantitative polymerase chain reaction (qPCR) chemistry, used as a routine method, on whole blood samples. A total of 755 clinical whole blood specimens were collected and tested simultaneously with TMA and qPCR assays. The data showed a qualitative agreement of 99.27% for positive quantified samples and 89.39% for those undetected between the two tested methods. Evaluation of viremia in positive samples highlighted a good correlation between TMA and qPCR chemistries in terms of International Units (ΔLog10 IU/mL: -0.29 ± 0.40). The TMA assay showed a significant correlation with qPCR in patients monitored for up to 3 months, thus allowing an accurate assessment of viremia in transplant patients. Therefore, TMA chemistry showed good agreement with qPCR testing, used as a current diagnostic routine. It also offers important advantages, such as FDA approval on plasma and In Vitro Diagnostic (IVD) on both plasma and whole blood, automated workflow with minimal hands-on time, and random access loading, thus enabling a rapid and reliable diagnostic in HCMV-infected patients.IMPORTANCEIn this paper, we describe the clinical performance of a novel transcription-mediated amplification (TMA) assay for the detection and quantification of human Cytomegalovirus (HCMV) DNA from whole blood samples. This is a pivotal analysis in immunocompromised patients [transplanted, HIV-positive, and Hematopoietic Stem Cell (HSC) recipients], and molecular tests with high sensitivity and specificity are necessary to evaluate the HCMV viral load in these patients. To our knowledge, this is the first in-depth evaluation of TMA chemistry for HCMV diagnosis on whole blood samples. Moreover, also technical aspects of this assay make it suitable for clinical diagnostics.