(Uploaded by Plazi for the Bat Literature Project) Background: Bats are among the most widely distributed mammals worldwide and can represent hosts or reservoirs for a number of different pathogens. Bartonella spp. are opportunistic bacterial pathogens, which are transmitted by a large variety of arthropods. The aim of this study was to investigate the presence and host-associations of these Gram-negative bacteria in heart tissues of bats collected in four different countries from eastern and central Europe and to analyze their phylogenetic relationship with other bat-associated bartonellae. Results: The results of this study show for the first time the presence of Bartonella spp. DNA in heart tissues of bats from central and eastern Europe. The overall prevalence of the infection was 1.38%. Phylogenetic analysis identified four new Bartonella spp. sequences, which were closely related with other Bartonella previously isolated from bats in Europe and North America. Conclusions: The gltA sequences of Bartonella spp. showed considerable heterogeneity in the phylogenetic analysis resulting in six different clades. Our study demonstrated the presence of Bartonella spp. only in heart tissues of bats from Romania, with two new bat species recorded as hosts (Myotis cf. alcathoe and Pipistrellus pipistrellus).