The prevalence of Bartonella spp. in wild rodents was studied in 19 geographical locations in Israel. One hundred and twelve rodents belonging to five species (Mus musculus, Rattus rattus, Microtus socialis, Acomys cahirinus and Apodemus sylvaticus) were included in the survey. In addition, 156 ectoparasites were collected from the rodents. Spleen sample from each rodent and the ectoparasites were examined for the presence of Bartonella DNA using high resolution melt (HRM) real-time PCR. The method was designed for the simultaneous detection and differentiation of eight Bartonella spp. according to the nucleotide variation in each of two gene fragments (rpoB and gltA) and the 16S-23S intergenic spacer (ITS) locus, using the same PCR protocol which allowed the simultaneous amplification of the three different loci. Bartonella DNA was detected in spleen samples of 19 out of 79 (24%) black rats (R. rattus) and in 1 of 4 (25%) Cairo spiny mice (A. cahirinus). In addition, 15 of 34 (44%) flea pools harbored Bartonella DNA. Only rat flea (Xenopsyla cheopis) pools collected from black rats (R. rattus) were positive for Bartonella DNA. The Bartonella sp. detected in spleen samples from black rats (R. rattus) was closely related to both B. tribocorum and B. elizabethae. The species detected in the Cairo spiny mouse (A. cahirinus) spleen sample was closely related to the zoonotic pathogen, B. elizabethae. These results indicate that Bartonella species are highly prevalent in suburban rodent populations and their ectoparasites in Israel. Further investigation of the prevalence and zoonotic potential of the Bartonella species detected in the black rats and the Cairo spiny mouse is warranted.