Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity.