The prevalence and genetic characteristics of Bartonella species in eastern bent-wing bats (Miniopterus fuliginosus) from Japan were investigated. Bartonella bacteria were isolated from 12/50 (24%) of bats examined. Analyses of sequence similarities of the citrate synthase gene (gltA) and RNA polymerase beta-subunit-encoding (rpoB) gene indicated that the isolates from M. fuliginosus were distinct from those present in known Bartonella species as the levels of similarity for both of the genes were lower than the cut-off values for species identification in Bartonella. A phylogenetic analysis of the gltA sequences revealed that the Miniopterus bat-associated strains fell into five genotypes (I to V). Though genotypes I to IV formed a clade with Bartonella from Miniopterus bats from Taiwan, genotype V made a monophyletic clade separate from other bat isolates. In a phylogenetic analysis with the concatenated sequences of the 16S rRNA, gltA, rpoB, cell division protein (ftsZ) gene, and riboflavin synthase gene (ribC), isolates belonging to genotypes I to IV clustered with Bartonella strains from Taiwanese Miniopterus bats, similar to the outcome of the phylogenetic analysis with gltA, whereas genotype V also made a monophyletic clade separate from other bat-associated Bartonella strains. The present study showed that M. fuliginosus in Japan harbor both genus Miniopterus-specific Bartonella suggesting to be specific to the bats in Japan. •The prevalence of Bartonella in Miniopterus fuliginosus was 24%(12/50).•M. fuliginosus in Japan harbored two novel Bartonella species in their blood.•One Bartonella species was the genus Miniopterus-specific Bartonella.•The other was distinct from other known bat-associated Bartonella.