Background and Objectives In Lao People's Democratic Republic, hepatitis B virus is highly endemic. However, blood donations are only screened for HBsAg, leaving a risk of transmission by HBsAg-negative occult infected donors. Here, we characterized first-time blood donors to assess prevalence of hepatitis B virus infections and occult infected donors. Materials and Methods Sera were screened for HBsAg, HBeAg and anti-HBs, anti-HBc and anti-HBe antibodies. Occult HBV infections (OBIs) were assessed in HBsAg-negative sera by PCR, and sera of HBsAg positive and occult infected donors were phylogenetically characterized. Results 96% of the donors were HBsAg positive, and 45.5% were positive for at least one of the hepatitis B virus serum markers. More than 40% HBsAg carriers were HBeAg positive, with HBeAg seroconversion occurring around 30years of age. Furthermore, 109% of HBsAg-negative, anti-HBc and/or anti-HBs-positive donors were occult infected with hepatitis B virus. Thus, at least 39% of blood donations would potentially be unsafe, but hepatitis B virus DNA copy numbers greatly varied between donors. Conclusion In Lao People's Democratic Republic, a sizable proportion of HBsAg-negative and anti-HBc antibody-positive blood donations are potentially DNA positive and infective for hepatitis B.