Cats are the main reservoirs of zoonotic Bartonella henselae which are the causative agents of Cat Scratch Disease (CSD). The aim of this study is to compare three diagnostic methods including culture followed by PCR from whole blood, nested-PCR from oral swab and whole blood, and IFA from serum samples. The diagnosis of B. henselae was compared with the bacteriological methods following conventional PCR and by two separate nested PCR from blood, and oral cavity swabs which were collected from 81 pet and stray cats in Istanbul, Turkey. Also the seroprevalence was determined by indirect fluorescent antibody (IFA) technique in the same animals. Bartonella spp. was determined in 26 (32%) of the blood samples by culture. Twenty of them were identified as B. henselae and 6 of them were B. clarridgeiae by following conventional PCR assay. Of 81 whole blood samples subjected to PCR, 29 (36%) were positive in the nested reaction. Of these, 20 were identified as B. henselae and 8 were B. clarridgeiae. However, one of the samples was found to be positive for both B. henselae and B. clarridgeiae DNA by the nested reaction. Of 81 oral swab samples subjected to PCR, 25 (31%) were positive in the nested reaction. Of these, 19 were identified as B. henselae and 6 were B. clarridgeiae. B. henselae IgG antibody seroprevalence was detected as 67% (54/81). Using the combination of blood and oral samples by Nested-PCR simultaneously may increase the sensitivity of the test. Also, the combination of the blood culture with nested-PCR and serology is likely to give the most definitive information in the diagnosis of bartonellosis in cats.