Bartonella henselae is the primary agent of cat scratch disease (CSD). In order to study the: genetic variation of B. henselae and the correlation of the various genotypes with epidemiological and clinical findings, two seminested, groEL- and pap31-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative Bartonella species. Compared to our reference intergenic spacer region-based PCR, the groEL- and pap31-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with B. henselae; neither Bartonella clarridgeiae nor other species were detected. Based on the groEL and pap31 sequences, B. henselae amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of B. henselae in patients with CSD.