The primary marker of current hepatitis B infection is the surface antigen (HBsAg), however HBsAg negativity does not exclude hepatitis B viremia. HBsAg variants can be responsible for such diagnostic failures. Here 13 different HBsAg variants were cloned, variant protein produced in a mammalian expression system, and tested using 7 commercial HBsAg diagnostic assays. Of 12 variants analyzed, 6 samples displayed similar reactivity to the positive control (containing standard HBsAg sequence) in most of the assays, but 6 samples, containing various mutations throughout the entire major hydrophilic region (MHR), showed reduced reactivity. It was found that the loss of cysteine at amino acid (aa) 124 in 1 sample affected the secretion as well as the reactivity of HBsAg in the expression system. Thus, not all assays are equally able to detect HBsAg variants, implying that, to attain an acceptable level of sensitivity, the antibody repertoire of the current assays should be extended.