Purpose: Epstein-Barr virus (EBV) infection is related to infectious mononucleosis or nasopharyngeal cancer, and its epidemiology may change according to the socioeconomic development of communities. This study aimed to evaluate the recent epidemiology of EBV seropositive rate in Korea. Methods: We retrospectively reviewed EBV serology test results obtained from a part of clinical care at Samsung Medical Center, Seoul, South Korea, from January 2000 to December 2017. Results: The EBV seropositive rate in 26,527 subjects during the study period was 81.0% (21,485/26,527): 44.4% (2,716/6,122) in subjects aged 0-9 years, 75.8% (2,077/2,739) in those aged 10-19 years, and 94.5% (16,692/17,666) in those aged ≥20 years. The EBV seropositive rate decreased from 89.4% (8,592/9,616) in 2000-2008 to 76.2% (12,893/16,911) in 2009-2017 (P<0.001). Especially, the EBV seropositive rate in subjects aged 0-19 years significantly decreased from 2000-2008 to 2009-2017 (0-9 years, 62.8% [1,172/1,866] in 2000-2008 and 36.3% [1,544/4,256] in 2009-2017; 10-19 years, 83.8% [745/858] in 2000-2008 and 70.8% (1,332/1,881) in 2009-2017) (P<0.001). Conclusions: The EBV seropositive rate in children has decreased in the last 20 years. As the age of patients with primary EBV infection increased, there is a need for interest in clinical manifestation, such as infectious mononucleosis, in adolescents and young adults.