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SUMMARYBartonellae are blood- and vector-borne Gram-negative bacteria, recognized as emerging pathogens. Whole-blood samples were collected from 58 free-ranging lions (Panthera leo) in South Africa and 17 cheetahs (Acinonyx jubatus) from Namibia. Blood samples were also collected from 11 cheetahs (more than once for some of them) at the San Diego Wildlife Safari Park. Bacteria were isolated from the blood of three (5%) lions, one (6%) Namibian cheetah and eight (73%) cheetahs from California. The lionBartonellaisolates were identified asB. henselae(two isolates) andB. koehleraesubsp.koehlerae. The Namibian cheetah strain was close but distinct from isolates from North American wild felids and clustered betweenB. henselaeandB. koehlerae. It should be considered as a new subspecies ofB. koehlerae. All the Californian semi-captive cheetah isolates were different fromB. henselaeorB. koehleraesubsp.koehleraeand from the Namibian cheetah isolate. They were also distinct from the strains isolated from Californian mountain lions (Felis concolor) and clustered with strains ofB. koehleraesubsp.bothieriisolated from free-ranging bobcats (Lynx rufus) in California. Therefore, it is likely that these captive cheetahs became infected by an indigenous strain for which bobcats are the natural reservoir.