There are few epidemiological and clinical studies about the presence of cat scratch disease (CSD) on the island of Crete. The objective of this study was to analyze a large number of patients with suspected CSD to define the frequency of Bartonella infections in Crete. From January 2005 to October 2008, we studied patients with suspected CSD from hospitals in Crete. Sera of the referred patients were tested by immunofluorescence assay (IFA). For some patients, we also received lymph nodes and blood samples that we tested for the presence of Bartonella henselae by molecular assays. Overall, we tested 507 serum samples and we found 56 (11%) cases of CSD. PCR assay was positive for 2 patients; one had a B. henselae positive lymph node and the other a positive whole blood sample. Significantly more CSD cases (62.5%, 35 of 56) were reported in children than in infants and adults (P<0.05). Moreover, we identified that most cases of CSD occurred between May and September (P=0.002) and December and January. CSD is prevalent in Crete and is mostly associated with an increase in outdoor activity.