Hepatitis B virus surface antigen (HBsAg) mutants occur in clinical specimens. We studied the analytic sensitivity and ability to detect recombinant and native mutants of 6 HBsAg assays. The ARCHITECT, AUSZYME MONOCLONAL, and AxSYM assays (Abbott Diagnostics, Abbott Park, IL); the ADVIA Centaur assay (Bayer Diagnostics, Tarrytown, NY); and the Test System 3 and VITROS ECi assays (Ortho Clinical Diagnostics, Raritan, NJ) showed comparable sensitivity with wild-type HBsAg. The ARCHITECT, AUSZYME, and AxSYM assays detected all mutants that were tested. The Test System 3 and VITROS ECi assays failed to detect mutants with amino acid substitutions at positions 143, 144, and 145, which are located in the immunodominant “a” determinant. The ADVIA Centaur failed to detect substitutions at position 145 and showed negative or very low positive results for substitutions at position 143. The inability to detect HBsAg mutants may lead to misdiagnosis of hepatitis B virus infection. Further studies on the prevalence of HBsAg mutants and the ability of commercial assays to detect them are needed.