The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Based on sequence divergence in the entire genome of )8%, HBV genomes have been classified into nine groups designated A to I. The genotypes of HBV have distinct geographic distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and the natural history of infection and response to antiviral therapy, further evaluations employing larger cohorts of patients are necessary to provide conclusive evidence. The analytical sensitivity of HBV surface antigen (HBsAg) and antibodies to the HBV surface antigen (anti-HBs) assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NATs) has so far been poorly investigated. Preliminary results show that new real-time NATs detect genotypes A to G with equal sensitivity. Different mechanisms intervening at the translational or post-translational level, including conformational changes, hydrophobic changes, insertion of basic residues, and reduced synthesis or secretion of HBsAg may account solely or in combination for escape mutations to the immune response and to detection in HBsAg immunoassays. The clinical significance of S gene mutants needs, similarly to that of HBV genotypes, to be investigated further. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is being continuously improved. Immunoassays based on polyclonal capture antibodies demonstrate the highest sensitivity for the recognition of recombinant mutants or serum samples