Although the genetic variability of hepatitis B virus (HBV) in HBV-infected patients has been extensively studied, reports on genotypes, subtypes and mutations in the S region of HBV strains from Chinese blood donors are limited. In this study, 245 blood samples from HBsAg-positive blood donors were collected from five geographically diverse blood centers in China. The S region of HBV was amplified, and the HBV genotype and subtype were determined. The amino acid sequences of the S region were aligned, and mutations related to the failure of immunization and HBsAg detection were determined. Of the 245 samples, 228 (93 %) were genotyped successfully. We found that genotypes B, C, D and A accounted for 58.8 %, 21.9 %, 6.6 % and 3.95 % of the isolates, respectively. The distribution of HBV antigen subtypes was as follows: adw (67.6 %), adr (23.3 %) and ayw (8.7 %). Mutations were present in 39 (17.1 %) of 228 samples in the major hydrophilic region (MHR) of the S region. This study demonstrated that HBV genotype/subtype B/adw was the most frequent strain circulating in HBV-infected Chinese blood donors, followed by C/adr. The occurrence of MHR mutants in HBV-infected blood donors and the potential failure to detect some of them in collected units poses a threat to transfusion safety.