The major circulating HIV-1 strains in Japan have been subtype B (B) followed by CRF01_AE (AE) in newly diagnosed HIV/AIDS cases. These two subtypes have distinct epidemiological characteristics; B predominates in men who have sex with men, while AE is observed mostly in heterosexuals engaging in high-risk sex. However, transmission networks of these two high-risk populations appear to be crossing over and diffusing. Here we report the emergence of previously unidentified HIV-1 AE/B recombinants in Japan. We initially identified 13 cases with discordant subtyping results with AE (gag MA)/B (pol PR-RT)/AE (env C2V3) by molecular phylogenetic analysis of 1,070 cases who visited Nagoya Medical Center from 1997 to 2012. Genetic characterization of full-length sequences demonstrated that they shared an identical recombinant structure, and was designated as CRF69_01B by the Los Alamos HIV National Laboratory. By reviewing gag, pol, and env sequences collected in the Japanese Drug Resistance HIV-1 Surveillance Network, we found five other CRF69_01B probable cases from different areas in Japan, suggesting that the strain is transmitted widely throughout the country. The time of the most recent common ancestor analyses estimated that CRF69_01B emerged between 1991 and 1995, soon after AE was introduced from neighboring countries in the mid-1990s. Understanding the current epidemic strains is important for the diagnosis and treatment of HIV/AIDS, as well as for the development of globally effective HIV vaccines.