Objective: Genotypes B and C are the prevalent hepatitis B virus (HBV) genotypes in eastern Asia. Although very rare in this region of the world, genotype D was found to be prevalent in a small area of western Japan. In this study, we confirm the frequency and clinical significance of co-infection with different genotypes among patients from that area infected with genotype D. Methods: Twenty-three patients from the same area of western Japan infected with HBV genotype D, determined using a genotyping enzyme immunoassay, were studied. Cloning was done using DNA extracted from serum samples, and polymerase chain reaction assays with the restriction fragment length polymorphism for HBV genotyping were performed with 10 clones from each patient. Results: Four (17.4%) of the 23 patients were found to be co-infected with HBV genotype C, and the HB surface antigen subtype was ayw in both mono- and co-infected patients. No clinical differences were found between mono-infected and co-infected patients carrying genotype D. Conclusion: A significant number of patients from the study area found to be infected with HBV genotype D were co-infected with genotype C. Additional study with a larger number of patients is needed to elucidate the possible clinical significance.