Bartonella is a genus of gram-negative bacteria that includes a variety of human and veterinary pathogens. These pathogens are transmitted from reservoirs to secondary hosts through the bite of arthropod vectors including lice and fleas. Once in the secondary host, the bacteria cause a variety of pathologies including cat-scratch disease, endocarditis, and myocarditis. Reservoirs of these bacteria are numerous and include several species of large mammals, mesocarnivores, and small mammals. Research on reservoirs of these bacteria has focused on western North America, Europe, and Asia, with little focus on the eastern and central United States. We assessed the prevalence of zoonotic Bartonella species among prairie-dwelling rodent species in the midwestern United States. Tissue samples (n = 700) were collected between 2015 and 2017 from five rodent species and screened for the presence of Bartonella DNA via PCR and sequencing of two loci using Bartonella-specific primers. Bartonella were prevalent among all five species, with 13-lined ground squirrels (Ictidomys tridecemlineatus) serving as a likely reservoir of the pathogen B. washoensis, and other rodents serving as reservoirs of the pathogens B. grahamii and B. vinsonii subsp. arupensis. These results demonstrate the value of studies of disease ecology in grassland systems, particularly in the context of habitat restoration and human–vector interactions.