The serological diagnosis hepatitis B (HBV) infections relies mainly on the detection of the small surface antigen (HBsAg). Immuno-enzymatic assays use antibodies directed against epitopes of the major hydrophilic region, in particular against a determinant. Genetic variants have been detected in vaccinated children, liver transplanted patients receiving anti-HBs immunoprophylaxis and chronic carriers with an occult infection (negative HBsAg). Substitutions in this region can induce conformational changes abolishing some diagnostic antibodies binding leading to false negative results. To estimate the prevalence of such substitutions, we studied 55 sequences of the gene S obtained between 2002 and 2004, carried out mainly in the search of changes of resistance to the lamivudine. The polymorphism of the major hydrophilic region (positions 102 to 169) was studied using Mutation Master software. Each sequence was compared with a consensus sequence of the same genotype. Genotype distribution is as follows: D, 40%; With, 20%; C, 16%; B, 11%; E, 9%; F, 2% and G, 2%. Changes I195 M and W196L, reflecting lamivudine resistance are present among 18 patients (33%). Substitutions of the major absorbent area are found among 31 patients (56%), of which a third associated with resistance mutations. The most frequent substitutions involved the following amino-acids: F/Y134 (7/31), M133 (6/31), D144 (6/31) and G145 (6/31). These two latter are known to be associated anti-HBs immunoglobulins or vaccine escape. BLOSUM scores are indicative of substitutions inducing important changes of physicochemical properties in 16 patients (29%). Such substitutions could thus potentially alter the binding of antibodies used in diagnostic assays. In conclusion, changes of the major hydrophilic loop of HBs antigen are not rare and could have an important implications for transfusion safety and diagnostic reliability.