We have previously reported 2 subtypes of hepatitis B virus (HBV) genotype B, one of which has the recombination with genotype C over the precore region plus core gene (Ba) and the other of which does not (Bj). A restriction fragment-length polymorphism method with 2 endonucleases was newly developed for distinguishing between subtypes Ba and Bj and was applied to 313 carriers of HBV genotype B in Japan. Subtype Ba was detected in 38 (12%) and subtype Bj in 275 (88%) of the carriers of HBV genotype B. Hepatitis B e antigen in serum was found more frequently in patients with chronic infection with subtype Ba than in those with chronic infection with subtype Bj (8 [32%] of 25 vs. 25 [9%] of 273; P<.01). The new method for distinguishing between Ba and Bj by restriction fragment-length polymorphism would be useful in examining the distribution of these 2 subtypes in situations in which HBV genotype B is prevalent.