Two sequential hepatitis B virus (HBV) strains obtained before and during an icteric flare-up of an occult HBV infection in a patient coinfected with human immunodeficiency virus revealed HBV surface antigen (HBsAg) test escape mutations, although the patient had never received hepatitis B-specific immunoglobulin. In contrast to the high HBV DNA loads, recurrence of HBsAg, and resulting icteric hepatitis, phenotypic analysis of the mutated HBV strains revealed significantly reduced replication efficacies in vitro, compared with wild-type HBV. Therefore, immune escape in the transiently anti-HBs-positive patient appeared to be crucial for persistence and reactivation. Immune escape mutants evolved even without exogenous selective pressure, hampered detection in HBsAg screening, and might be transmitted during reactivation with high HBV loads.