Objective: To investigate the prevalence of hepatitis B virus (HBV) infection and to diagnose an occult HBV infection in healthy Thai subjects after implementation of the expanded program on immunization (EPI) in newborns.Material and Method: The detection of HBsAg, anti-HBs and anti-HBc was done from serum samples of 5,886 healthy first year students from Huachiew Chalermprakiet University collected between 2009-2011 by immunochro-matography rapid assay (Alcon, USA). In case of only anti-HBc positive, the results were confirmed with chemiluminescence enzyme immunoassay (EIA) method and then they were investigated further for HBV occult infection by nested polymerase chain reaction technique.Results: A total of 78% (4,593/5,886) healthy first year students who had been vaccinated with HBV vaccine since birth were found to have no HBV markers, while 18.4% (1,083/5,886) had only anti-HBs. The prevalence of infection was 3.6% (210/5,886). Identification of patterns of HBV infection among the 210 infected subjects found that (1) Immuned due to past infection was 61.9% (130/210) (2) Infected with hepatitis B virus was 38.1% (80/120). The prevalence of both HBsAg and anti-HBc was 28.6% (60/210), while the prevalences of HBsAg and Anti-HBc alone were 3.8% (8/210) and 5.7% (12/210), respectively. Anti-HBc alone group was repeated with chemiluminescence EIA and HBV DNA was negative.Conclusion: The prevalence of HBV infection was 3.6% (210/5,886), however, seropositive rate of HBV infection was 1.4% (80/5,886). Most antibodies from vaccination had substantially declined to the point that it was undetectable. Therefore, the overall study showed an effective implementation of EPI in newborns.