Bartonella species are pathogens of emerging and reemerging significance, causing a wide array of clinical syndromes ranging from cat scratch disease and persistent bacteremia to disseminated bacillary angiomatosis, peliosis, endocarditis, trench fever, encephalitis, and neuroretinitis. Although improved blood culture techniques were a primary factor in the discovery of Bartonella endocarditis, isolation of the organism still requires prolonged periods and specific growth conditions. Thus, serologic testing for Bartonella henselae infections has become the cornerstone of clinical diagnosis, replacing the skin test. Molecular techniques, including polymerase chain reaction, offer high sensitivity and specificity, rapid availability of information, and the ability to differentiate Bartonella organisms at the highest level. This article focuses on the use of microbiologic, serologic, and molecular means to aid in the clinical diagnosis of Bartonella infections.