Epstein-Barr virus (EBV) is an extremely common herpesvirus that may cause asymptomatic infection or various diseases, including infectious mononucleosis, certain lymphoproliferative diseases and several types of neoplasms. Vaccine development is an important strategy to reduce the burden of EBV-associated diseases and the timing of vaccinations should be before primary infection occurs. In the past, more than 90% of Thai children were infected with EBV in early childhood. Now, due to the improved healthcare system in Thailand, we aim to determine current prevalence of EBV infection among people in different age groups. A total of 538 sera were collected from residents of Khon Kaen province in northeastern Thailand for detecting anti-EBV IgG. Sera of infants under 2-years-old were also tested for anti-EBV IgM and EBV-DNA. The prevalence of anti-EBV IgG was 47.1% (95% CI: 23.3-70.8) in infants aged 0-6 months, 34.9% (95% CI: 23.1-46.7) in those aged 6-24 months, 87.9% (95% CI: 79.5-96.3) in children aged 3-5 years and then maintained at above 95% through adulthood. These seropositivity rates among Thai children remain similar to those found in a previous study conducted 20 years ago. Thai children are still exposed to EBV from an early age. Therefore, a prophylactic vaccine should be given within the first two years of life.