Bacteria of the genus Bartonella are emerging zoonotic bacteria recognized in a variety of human diseases. Due to their poor chemical reactivity, these fastidious bacteria are poorly characterized using routine phenotypic laboratory tests. Identification is usually achieved using molecular techniques that are time-consuming, expensive and technically demanding. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a new technique for bacterial species identification. This study evaluated the use of MALDI-TOF MS for rapid genus and species identification of Bartonella species. Reference strains representing 17 recognized Bartonella species were studied. For each species, MS spectra for four colonies were analysed. The consensus spectrum obtained for each species was unique among spectra obtained for 2843 bacteria within the Bruker database, including 109 alphaproteobacteria. Thirty-nine additional blind-coded Bartonella strains were correctly identified at the species level, including 36 with a significant score. Altogether, these data demonstrate that MS is an accurate and reproducible tool for rapid and inexpensive identification of Bartonella species.