BACKGROUND: Primary EBV infection is common in childhood, and dual positivity of serum Epstein-Barr virus (EBV) IgM and Cytomegalovirus (CMV) IgM antibodies occurs in some cases of primary EBV infection in children. The aim of this study was to evaluate the cause of EBV and CMV IgM dual positivity whether it is a false positive finding or a true coinfection of EBV and CMV. METHODS: A total of 494 pediatric patients aged 18 years or less diagnosed with primary EBV infection manifesting as infectious mononucleosis, in Seoul National University Bundang Hospital from March 2004 through February 2016, were recruited. The diagnosis of primary EBV infection was based on a positive EBV viral capsid antigen (VCA) IgM antibody, and serum CMV IgM antibody and liver enzymes was checked at the same time in 149 subjects. When serum CMV IgM is positive, additional tests to confirm the diagnosis of CMV infection were evaluated. RESULTS: Of 149 children with primary EBV infection children, 40 (26.8%) revealed serum CMV IgM dual positivity along with serum EBV VCA IgM positivity. True CMV infection was confirmed only in one child of 40 (2.5%) who was positive for serum CMV Ag and urine CMV PCR and negative for serum CMV IgG antibody. In the other 39 children, serum CMV Ag and urine CMV PCR or cultures were all negative at the time of serum CMV IgM and EBV VCA IgM dual positivity. Dual positivity were higher in infants and lower in adolescents in children with primary EBV infection (p = 0.013). Laboratory markers of hepatic involvement (liver enzymes) were significantly elevated in children with dual positivity than those with negative CMV IgM (p = 0.026), correlating with serum EBV and CMV IgM titers. CONCLUSIONS: Serum EBV VCA IgM and CMV IgM dual positivity is more prevalent in children with primary EBV infection than reported before. Our results indicate that this dual positivity is a false positive finding, possibly due to antigenic cross-reactivity, rather than coinfection with CMV.