To control hepatitis B virus (HBV) infection, a universal HBV vaccination program for infants was launched in Taiwan in 1984. The aim of this study was to investigate the role of B-cell and T-cell epitope variations of HBsAg and polymerase in HBV infection in vaccinated children. One hundred sixty-three sera from vaccinated children were enrolled randomly. HBV serum markers, including hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) and core antigen (anti-HBc), were detected by ELISA. Nucleotide sequences encoding the S and the pre-S regions of HBsAg were analyzed in all HBsAg positive sera. Five children were HBsAg positive. Sequence analysis of S, pre-S, and overlapped polymerase (P) genes showed that HBV isolates of HBsAg-positive vaccinees were variants; no G145R but G145A and other substitutions were found in the "a" determinant. Fifteen, six, and eight amino acid substitutions within B-cell and T-cell epitopes of S, pre-S, and P regions were detected, respectively. Several immune-epitope mutants, such as S45T/A, N131T, I194V, and S207N in S, were detected in all isolates. In conclusion, our results suggested that these naturally occurring immunoepitope mutants, which changed their immunogenicity leading to escape from immune response, might cause HBV infection.