OBJECTIVE To explore the variations of gene S in hepatitis B viruses of hepatitis B patients and provide experimental evidences for the mutation analysis of viral gene. METHODS The virus DNA load in hepatitis B patient donors was detected by real-time polymerase chain reaction (PCR) and gene sequence analysis. And a comparison was made with standard strain by the software DNAstar. RESULTS (1) Gene S was successfully amplified and sequenced in 15 hepatitis B patients. Three samples had I→T mutation at residue 126 in HBsAg "a" antigenic determinant. (2) Sixteen hepatitis B patients had 67 nucleotide mutations, including 14 residues in PreS1 and 6 residues in PreS2. Mutations nt 3036 T→C, nt 3039 T→G, nt 3066 C→T and L88V existed in PreS1 gene in all samples. CONCLUSIONS HBV genome is susceptible to nucleotide mutations. Some residues have geographically restricted mutations in gene S region. And understanding the significance of these mutations may help clarify the pathogenesis of hepatitis B and provide new experimental evidence for its gene diagnosis and prevention.