AbstractBased on molecular data, previous studies have suggested a high overall diversity and co‐infection rates of Bartonella bacteria in wild rodents and their fleas. However, partial genetic characterization of uncultured co‐infecting bacteria limited sound conclusions concerning intra‐ and inter‐specific diversity of the circulating Bartonella. To overcome this limitation, Bartonella infections of wild populations of two sympatric gerbil species and their fleas were explored by multiple isolations of Bartonella organisms. Accordingly, 448 pure Bartonella isolates, obtained from 20 rodent blood and 39 flea samples, were genetically characterized to the genotype and species levels. Results revealed a remarkable diversity and co‐infection rates of Bartonella among these sympatric rodents and their associated fleas. Specifically, 38 genotypes, classified into four main Bartonella species, were identified. Co‐infection was confirmed in 56% of the samples, which contained two to four Bartonella genotypes per sample, belonging to up to three different species. Recombination within and between these species was demonstrated, serving as a direct evidence of the frequent bacteria–bacteria interactions. Moreover, despite the noticeable interchange of common Bartonella genotypes between rodents and fleas, the co‐occurrence of genotypes was not random and differences in the overall diversity, and the ecological and phylogenetic similarities of the infection compositions were significantly associated with the carrier type (rodent vs. flea) and the rodent species. Thus, comprehensive identification of the co‐infecting organisms enabled the elucidation of ecological factors affecting the Bartonella distribution among reservoirs and vectors. This study may serve as a model for the investigation of other vector‐borne organisms and their relationships with Bartonella.