Two considerations led us to study the genetic diversity and origin of hepatitis B virus (HBV) in Dutch blood donors. Firstly, an HBV-infected Dutch blood donor was found negative by four assays used commonly for detection of HBV surface antigen (HBsAg). How variable is HBsAg among HBV infected blood donors? Secondly, the WHO recommends universal vaccination against HBV, but north-west European countries limit vaccination to groups at risk of HBV. This policy may reduce hepatitis B among low-risk, unvaccinated persons if HBV strains that infect low-risk persons stem from local at-risk groups. Studying the nucleotide sequence of the S-gene of HBV from 63 Dutch blood donors, considerable variation was found. The majority of the donor strains (52/63, 83%) appears closely related to local HBV isolates as present in intravenous drug users, immigrants, and homosexual men. The remaining 11 (17%) HBV strains belong to various non-Western genotypes. This implies that an indigenous Dutch HBV strain (heterosexually transmitted, not associated with intravenous drug abuse, or immigrants) does not exist, and it supports the policy in low endemic countries to limit vaccination to at-risk groups. On the other hand, it must be realised that, after 20 years of vaccination of at-risk groups, HBV still circulates in the at-risk groups and Dutch blood donors acquire the HBV strains involved.