At present in Japan, only high-risk infants born to chronic hepatitis B virus (HBV)-infected mothers are given HBV vaccine. However, children can contract the virus from other HBV-infected family members, including fathers. The aim of this study is to present substantial and unequivocal evidence of father-to-child transmission of HBV infection using techniques including homology analysis and phylogenetic analysis. Thirteen chronic HBV-infected members of five families that included eight children and their respective fathers were enrolled in this study. Homology analysis and phylogenetic analyses of 2 coding region, the S gene and X gene, from the HBV genome were performed comparing the 13 nucleotide sequences from the 13 subjects. The nucleotide homology among the five sets of fathers and children was quite high (99.3-100%). A phylogenetic tree constructed on the 13 nucleotide sequences showed that all 5 sets of fathers and children were grouped into the same cluster with high bootstrap values. These results strongly indicate that father-to-child transmission is an important route of HBV infection in Japan and it is recommend that universal vaccination against HBV infection be instituted immediately in Japan for all children, in accordance with the WHO recommendation of 1997.