This chapter describes the methodology, analysis, and assessment of the significance of hepatitis B virus (HBV) mutations selected during antiviral therapy. Included in this description is the methodology for genotype classification. The major area of sequence-analysis methodology focuses on the reverse transcriptase region of the polymerase gene, in particular the catalytic domains A–E. This region coincides with the “a” determinant of the S gene in the overlapping reading frame. Other clinically significant HBV mutations, which are located in other genes and regulatory regions such as the basal core promoter and precore gene [(both of which are associated with loss or reduction in the production of hepatitis Be antigen (HBeAg)], may also be identified. Multiple mutations within a gene and the combination of mutations from other areas of the genome may act in a compensatory manner, altering the resistance pattern, replication phenotype, and pathogenesis profile of viral infection. Thus, the crosslinking of genomic data has potentially important implications for patient management.