Heterosexual contact is one of the common routes of transmission for hepatitis B virus (HBV) among adults in Taiwan, but only a few studies have provided direct evidence at the level of the HBV genome of infected couples with acute non-fulminant hepatitis to document a common source. By cloning and sequencing polymerase chain reaction-amplified HBV-DNA, we analysed the sequences of the conserved region of the surface gene (nucleotide (nt) 305-513, representing 6.5% of the viral genome) of HBV in five HBV-infected index patients, their spouses and four randomly selected HBV carriers as controls. Risk factors associated with acute HBV infection in index cases were sexual contact with their spouses within 3 months before the onset of hepatitis. For all five couples, the HBV-infected index patient and the spouse shared a 100% sequence homology of HBV-DNA. In contrast, there was significantly more variation (mean heterogeneity 6.1%, range 1-13.9%) in the amplified region between the five couples and between each couple and the controls (P < 0.001). This study demonstrated that sequence analyses can correlate well with epidemiological findings and confirm the value of the molecular approach for linked infections of HBV through heterosexual contact between spouses. Susceptible adults should receive vaccination.