Abstract Background Cytomegalovirus (CMV) is a DNA virus that often causes severe disease in immunosuppressed or immunocompromised patients. With the expanding indications for immune-modulating agents and transplant patients, the number of patients at risk for developing CMV disease is increasing. Test results are used to determine active CMV disease, risk of developing active disease, response to therapy, risk of relapsed infection, and/or appropriate time to discontinue therapy. For this purpose, molecular assays are attractive because of their broad linear range and low limits of detection. CerTest Biotec has developed a quantitative real time PCR assay for the detection and quantification of cytomegalovirus in plasma samples. Methods Plasma-EDTA samples characterized as positive or negative and quantified by IU/ml were selected. The selected samples were collected from both male and female adults (except 1 sample of an infant) between July 2016 and December 2020. Two CE-marked molecular reference assays were used for the comparative analysis: RealStar® CMV PCR Kit 1.0 (Altona), using the CFX96™ Real-Time PCR Detection System (Bio-Rad) and extracted with the QIAamp® MinElute® Virus Spin Kit (QIAGEN); and cobas® CMV (Roche Diagnostics), using the cobas® 8800 system. VIASURE analysis was performed using the following workflow: the automated extraction method MagDEA® Dx SV with magLEAD® 12gC and CFX96™ Real-Time PCR Detection System (Bio-Rad). Results A total of 273 EDTA-plasma clinical samples were analysed, 120 samples were reported as CMV positive samples and quantified and 153 samples were reported as CMV negative samples. Comparative analysis determined >99% sensitivity and specificity. Regarding quantification, all methods obtained a correlation value of R2 ≥ 0.77. Conclusion With this study it was possible to observe that the VIASURE CMV q Real Time PCR Detection Kit present good clinical sensitivity and specificity compared to reference molecular assays results. No false positive results were observed. However, all variables employed on the workflow contribute to final quantification results and therefore should be taken into account as accumulative error in the calculation.