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A number of Bartonella isolates were obtained from seven species of rodents sampled from 12 geographic sites representing the major biotic communities of the southeastern United States. Bartonella were isolated from the blood of 42.2% of 279 tested rodents. The highest prevalence of infection typically occurred among the most commonly captured species in the rodent community. Four phylogenetic groups, uniting 14 genotypic variants of Bartonella, were identified by sequence analysis of the citrate synthase gene. The level of sequence homology between genotypic groups varied from 88.8% to 96.4%, and the degree of homology among variants within groups was ≥ 97%. Cotton rats (Sigmodon hispidus) harbored up to three phylogenetic groups of Bartonella at a single site, and Bartonella of two phylogenetic groups were isolated from a single rodent. All the Bartonella isolated from three species of Peromyscus clustered in a single distinct phylogenetic group, suggesting some host specificity may occur. Mouse ascitic fluids produced in BALB/c mice inoculated with Bartonella of three phylogenetic groups demonstrated high indirect fluorescent antibody (IFA) titers to homologous antigens. However, use of eight Bartonella antigens in an IFA test with sera from 394 wild-caught rodents resulted in either little or extremely low titers of antibody.