Epstein-Barr virus (EBV) is a ubiquitous virus that establishes a latent infection within the host and in some cases can lead to the development of EBV- Associated lymphomas, lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, solid tumors, and other diseases. We studied the clinical significance of detecting EBV DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of 2146 patients who had blood specimens sent to the Johns Hopkins Hospital clinical laboratory for viral quantitative real- Time polymerase chain reaction assay over a 5-year period. Within this largely immunocompromised and hospitalized cohort, 535 patients (25%) had EBV detected in plasma or PBMCs.WhenEBVwas detected in the absence of anEBV+ disease (n5402), it was present only in PBMCs in 69% of cases. Immunocompromised patients were less likely to have EBV in plasma than in PBMCs in the absence of EBV+ disease. In patients withactive, systemicEBV1diseases(n5105),EBVwasdetectedin plasma in 99%of cases but detected inPBMCsin only 54%. Across a range of copy number cutoffs,EBVin plasma had higher specificity and sensitivity for EBV+ disease as compared with EBV in PBMCs. EBV copy number in plasma distinguished untreated, EBV+ lymphoma from EBV1 lymphoma in remission and EBV- lymphoma, and also distinguished untreated, EBV+ posttransplantation lymphoproliferative disorder (PTLD) from EBV+ PTLD in remission and EBV- PTLD. EBV copy number quantification is a useful diagnostic marker across the spectrum of EBV+ diseases, even among immunocompromised patients, with plasma specimens more indicative of EBV+ disease than PBMCs.