Background: Immune escape mutation is a major reason for vaccination failure in vertical transmission of hepatitis B virus (HBV). Objectives: The aim of the current study was to explore the characteristics of HBV S region gene in vertical transmission and the influence of mutations on vaccination failure in HBV S region gene. Methods: A total of nine mother-child pairs with chronic HBV were recruited in this study, and all of the children received the vaccine. Next-generation sequencing (NGS) was used to investigate the sequences of HBV S gene from the mother and children. Results: The analysis of the sequencing data showed that all mother-child pairs are genotype B and genotype C mixed infection and the dominant genotype did not change in vertical transmission, and the consensus sequences of mothers and children were highly similar. Furthermore, the diversity of most amino acid sites had a small difference between mothers and children, and the mutations shared by mothers and children made up a high proportion of children's mutations; the rate was 83.13% ± 8.45%. Also, the research found many mutations that could cause immune escape in major hydrophilic region. Especially in "a" determinant, sC137S or sC139Y, which had an important influence on the structure of "a" determinant, were observed in all mother-child pairs. Conclusions: All the above results indicated that HBV S gene was highly conserved in vertical transmission, and these immune escape mutations that pre-exist in mothers were transmitted to children and resulted in vaccination failure.