Objectives: We investigated the prevalence of hepatitis B virus (HBV) markers and occult HBV infection in a general adult population in Korea. Methods: HBsAg, anti-HBs and anti-HBc were analyzed on 1,091 samples of routine medical check-up examinees by chemiluminescence immunoassay. Nucleic acid amplification test (NAT) was performed on 1,047 HBsAg-negative samples by multiplex real-time polymerase chain reaction (PCR) kit for simultaneous detection of HBV, hepatitis C virus, and human immunodeficiency virus (Cobas Taqscreen MPX) in pools of six and reactive pools were resolved to individual samples, and further discriminated by PCR-based assay for HBV (Cobas Ampliscreen HBV). Results: The prevalences of HBsAg, anti-HBc, and anti-HBs were 4.0, 39.3, and 75.4%, respectively. The prevalence of anti-HBc significantly decreased with decreasing age (p< 0.001). Occult HBV infection was found in 7 (0.7%) of 1,047 HBsAg-negative subjects, and 5 of them were anti-HBc-negative. Sequencing of HBV S gene in 3 cases revealed one wild-type and two mutant strains (W74S, F85Y; T63I, W74S, T131N substitutions). Conclusions: This study helps to understand the current status of hepatitis B infection and the prevalence of occult HBV infection in a general adult population in Korea.