With the improvements in diagnostic techniques, Bartonella henselae (B. henselae) infection has recently been recognized to cause a widening spectrum of diseases. Cats are the natural reservoir hosts of B. henselae. The current study aims to investigate the prevalence of B. henselae infection in the cat populations in China. Polymerase chain reaction (PCR) and bacterial cultures confirm that 12.7% of the tested cats were positive for the infection. Old age and outdoor exposure were statistically associated with the infection. Multilocus sequence typing and eBURST analysis of the cat isolates collected in the present study show that 65.4% of the isolates belong to sequence type 1 (ST1). Three new STs (ST16–18) were identified in Midwestern China. These results may aid our understanding of the population structure of B. henselae in China and the relationship between human and cat strains in subsequent studies. Author Summary: Bartonella infection (Bartonella henselae in paticular) is responsible for a widening spectrum of human diseases. Cats are thought as the major source of human infections. In most of the developing countries, however, this infection lacks recognition especially in clinicians. Some well known bartonelloses, like cat-scratch disease, bacillary angiomatosis, and infective endocarditis had not been clinically noticed in these countries. In the present study, the prevalence of Bartonella infection in cat populations was investigated in mainland China. As a result, 12.7% of cats were confirmed positive with Bartonella infection. Factors of age, gender, outdoor experience, and geographic location were analyzed in association with infection. The authors found old cats and stray cats are susceptible populations. Some new molecular types are prevalent in Mid-Western China. Inclusion of these types also changed the characteristics of original molecular structure. This research provides the first molecular investigation of Bartonella infection in cats in China, and promotes our understanding of the relationship between human and cat strains in subsequent studies. [ABSTRACT FROM AUTHOR]