Clinicians use molecular tests to detect Herpesviridae from blood without fully appreciating limitations of testing. Studies are needed to enhance our understanding of the impact of Herpesviridae latency on molecular testing. We retrospectively performed quantitative Epstein-Barr virus (EBV) on sera from patients between the ages of 1 and 30 who demonstrated serologic evidence of acute EBV (n = 50) or remote EBV (n = 50) infection. Epstein-Barr virus DNA was detected in 70% of acutely infected and 4% of remotely infected patients. Sera from acutely infected patients had higher EBV copy number than convalescent sera. Our results suggest that serology should be performed as the initial diagnostic test for acute EBV. The role for polymerase chain reaction in immunocompromised patients with impaired antibody responses or as a 2nd-line diagnostic test when serologic results are equivocal deserves further study.