Prevalence of Bartonella infection among 275 cats in 9 sites from 4 geographical regions (northern area: Chiang Mai; central area: Kanchanaburi, Ratchaburi, and Bangkok; northeastern area: Khon Kaen, Roi Et, Ubon Ratcharthani, and Nakhonratchasima; southern area: Songkhla) of Thailand was investigated. Overall, Bartonella species were isolated from 27.6% (76 of 275) of the cats. The isolation rate varied from 12.8% (5 of 39) in Songkhla (southern area) to 50.0% (26 of 52) in Khon Kaen (northeastern area). Bartonella henselae and B. clarridgeiae were isolated from 82.9% (63 of 76) and 11.8% (9 of 76) of the Bartonella-positive cats, respectively. Coinfection with both species was found in 5.3% (4 of 76) of the bacteremic cats. Of the 67 bacteremic cats from which B. henselae was isolated, 48 (71.6%) and 13 (19.4%) were infected with only Type I and Type II, respectively. Coinfection with both types was observed in 9.0% (6 of 67) of the B. henselae-positive cats. To our knowledge, this is the first report on the presence of Bartonella infection in domestic cats from Thailand, which constitute a large reservoir of Bartonella infection in this country.