There are 7 genotypes of hepatitis B virus (HBV). Whether superinfection of HBV carriers with different HBV genotypes occurs remains unknown. We therefore determined the HBV genotype and association between superinfection and acute exacerbation of disease in a cohort of 244 patients with chronic HBV infection who had elevated serum aminotransferase levels for at least 1 year. Within this group, 103 patients experienced acute exacerbation with an annual incidence of 13%, and 20 of the 103 patients had IgM antibody to hepatitis B core antigen (IgM anti-HBc). These 20 patients had a higher prevalence of genotype C infection (65%) than the remaining 83 anti-core IgM–negative patients (40%) who also had acute exacerbations (P < .05). Detailed analysis of HBV genotypes and sequences of the variable pre-S gene were determined in serial samples from 20 patients with IgM anti-HBc–positive acute exacerbations (group A), 20 patients with IgM anti-HBc–negative acute exacerbations (group B), and 20 patients without exacerbations (group C). Two (10%) of the group A patients had virologic evidence of HBV superinfection during acute exacerbation, one superinfected with heterotypic virus and the other with homotypic virus. The newly introduced virus disappeared after the exacerbation and the original virus resumed thereafter. The calculated prevalence of HBV superinfection in the hepatitis B carriers and those with acute exacerbations was 0.8% (2 of 244) and 1.9% (2 of 103), respectively. In conclusion, superinfection of HBV on hepatitis B carriers indeed occurs and may cause acute exacerbations, albeit at a low frequency even in hyperendemic areas of HBV infection.