Two novel mutants of the hepatitis B virus Surface antigen (HBsAg) were characterized. The mutants were isolated from asymptomatic patients who were found to be persistently positive for both I-BsAg and anti-hepatitis B surface antibody (anti-HBs) and a long-lasting anti-HBc (core) IgM. Polymerase chain reaction (PCR) and sequencing were performed to surface antigen region and revealed two genotype D mutants 1--1BV. Aligned with known I-BsAg sequences from GenBank, the mutant variants matched to consensus with eight distinct genotypes (A to H) of hepatitis B virus. Multiple sequence alignment provided a more sensible way of detecting sequence homology and identifying the HBV isolates. From this alignment, certain positions were highly conserved with the previously identified and recorded Egyptian isolates, while other positions were not. The importance of the two variants lies in the observation that mutations in the surface antigen coding region can change the immunodominant region structure and therefore alter the group specific determinant antigenicity. Phylogenetic tree based on I-BsAg partial nucleotide sequence was displayed. In conclusion, by understanding the HBsAg major immunodominant region nucleotide sequence, highly sensitive diagnostic assays can be achieved for detection of HBV in clinical specimens. Control of these EBV mutants, which will require new drugs, vaccines, and treatment strategies, will become the next major challenge on the path to eventual elimination of HBV infection. Keywords: HBsAg, PCR, Sequencing, Phylogenetic.