Quantitative detection of cytomegalovirus (CMV) DNAemia by real-time PCR is currently the primary choice for the surveillance of active CMV infection in allogeneic stem cell transplant (Allo-SCT) recipients. Nevertheless, no universally accepted standards for CMV viral load quantitation are available, this being critical when clinical studies involving various participant centers that use different assays are planned.To compare the analytical performance of two commercially-available real-time PCR assays carried out at two different centers.Plasma samples were collected at the University Hospital Virgen del Rocío (A) and at the Hospital Clínico Universitario (B) and were exchanged for analysis. In hospital A, DNA was extracted manually and viral loads were quantitated with the Affigene CMV Trender. In hospital B, DNA extractions were performed using an automated system and viral loads were quantitated using the CMV PCR Kit manufactured for Abbott by Qiagen.A total of 80 samples obtained from Allo-SCT recipients (20 samples per each of the following CMV DNA load groups: undetectable level, 10,000 copies/mL) were analyzed. The Affigene CMV Trender assay yielded significantly higher viral loads than the Abbott CMV real-time PCR Kit, regardless of the DNA extraction method employed.Automated DNA extraction systems should be thoroughly evaluated for their analytical performance. Local guidelines for the initiation of pre-emptive therapy based on commercial real-time PCR assays measurements must be established as long as universally accepted standards for quantitative analysis of CMV DNAemia are not available.