The prevalence of occult hepatitis B virus infection (OBI) among people with a family history of chronic hepatitis B virus (HBV) infection is unclear. Serum samples were collected from 747 hepatitis B surface antigen (HBsAg)-negative people with a family history of HBV infection and 579 HBsAg-negative volunteer blood donors. The presence of HBV DNA was evaluated using nested PCR with primers specific for the X, S, and C regions of HBV. The Pre-S1/Pre-S2/ S region PCR products for the OBI group and their family members with chronic HBV infection (control group) were sequenced and compared. The prevalence of OBI was 8.0% (60/747) among HBsAg-negative people with a family history of chronic HBV infection, compared to 2.6% (15/579) among the blood donors (P<0.05). The prevalence of HBV genotype B infection was lower in the OBI group than in the control group (P=0.031). The substitution rates in the major hydrophilic region and the a determinant seemed to be higher in the OBI group (0.893 vs. 0.507; 1.042 vs. 0.403, respectively), and stop codon mutations more frequent in the OBI sequences (OBI: 2/26, 7.7% vs. control: 0/31, 0%). However, none of these differences was statistically significant (P=0.237, 0.199, 0.201, respectively). In summary, the prevalence of OBI among HBsAg-negative people with a family history of chronic HBV infection was significantly higher than that in Chinese blood donors. However, S region mutations and the escape mechanism are not likely to be the major causes of increased prevalence of OBI. J. Med. Virol. 87:1890-1898, 2015. (c) 2015 Wiley Periodicals, Inc.