The present study aimed to detect Bartonella DNA in cats belonging to shelters, and to evaluate risk factors, clinical signs, and hematological abnormalities associated with infection. Complete blood counts and screening for the presence of Bartonella DNA were performed on cats' ethylenediamine tetraacetic acid anticoagulant-blood samples. Eighty-three cats (39.9%) were positive for Bartonella species. Bartonella DNA was also detected in fleas and in the blood of cats infested by positive flea. Cats that had not been sterilized, had outdoor access, had histories of fights, and had concurrent flea infestation were more likely to be infected by Bartonella species (P < 0.05). Age and sex were not associated with infection. Fifty-one (38.6%) symptomatic cats were positive to Bartonella species (P > 0.05). Clinical conditions most commonly observed were signs of respiratory abnormality and Sporothrix species coinfection (P > 0.05). Regarding hematological changes, eosinophilia was associated with infection (P < 0.05). A high frequency of Bartonella species infection was found in shelter cats and highlights the importance of adequate flea-control programs to prevent infection in cats and consequently in adopters and other animals.