BACKGROUND: The Epstein-Barr virus (EBV) is one of the most common viruses found in humans, causing lifelong infection in up to 95% of the world population. OBJECTIVES: To analyze the seroprevalence of EBV infection in different population groups in Croatia. METHODS: During a 2 year period (2015-2016), a total of 2022 consecutive serum samples collected from Croatian residents were tested for the presence of EBV-specific viral capsid antigen (VCA) immunoglobulin M (IgM) and IgG antibodies using an enzyme-linked immunoassay. IgM/IgG-positive samples were further tested for IgG avidity. RESULTS: The overall prevalence of EBV IgG antibodies was 91.4%. Females had significantly higher IgG seroprevalence than males (93.1% vs. 89.9%, P = 0.008). According to age, IgG seropositivity increased progressively from 59.6% in children age < 9 years to 98.3% in 30-39 year olds, and remained stable thereafter (P < 0.001). The IgG seroprevalence differed significantly among groups: 68.1% in children/adolescents and 95.9% in adults ; multiple sclerosis (100%), hemodialysis patients (97.7%), heart transplant recipients (93.8%), hematological malignancies (91.2%), and Crohn's disease (88.5%), P < 0.001. IgM antibodies were detected in 9% of participants. Using IgG avidity, recent primary EBV infection was documented in 83.8% of IgM- positive subjects < 9 years old, 69.2% age 10- 19, 33.3% age 20-29, and 3.6-4.2% > 40. All IgM positive participants > 40 years showed high IgG avidity. Logistic regression showed that age is associated with EBV IgG seropositivity. CONCLUSIONS: EBV is widespread in the Croatian population. Older age appears to be the main risk factor for EBV seropositivity.