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 eight groups designated A to H. The genotypes of HBV have distinct geographical distributions. Although preliminary clinical studies seem to indicate that there is an association between HBV genotype and natural history of infection and response to antiviral therapy, further evaluations on larger collectives of patients are necessary to give a clearer picture of the subject. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype. The influence of genotypic variability on the sensitivity of nucleic acid amplification tests (NAT) has so far been poorly investigated. Preliminary results show that new real-time NAT detect genotypes A to G with an 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 conjunction for escape mutations to the immune response and to detection in HBsAg immunassays. The clinical significance of S-gene mutants, needs in analogy to that of HBV genotypes, to be further investigated. HBV mutants are stable over time and can be transmitted horizontally or vertically. The sensitivity of HBsAg assays for mutant detection is continuously improved. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S-gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants and strategies for the (differential) screening of mutants need to be developed and evaluated.