Three novel isolates of the genus Bartonella were recovered from the blood of two patients enrolled in a clinical trial for the treatment of chronic stage Bartonella bacilliformis infection (verruga peruana) in Caraz, Ancash, Peru. The isolates were initially characterized by sequencing a fragment of the gltA gene, and found to be disparate from B. bacilliformis. The isolates were further characterized using phenotypic and genotypic methods, and found to be genetically identical to each other for the genes assessed, but distinct from any known species of the genus Bartonella, including the closest relative B. bacilliformis. Other characteristics of the isolates, including their morphology, microscopic and biochemical properties, and growth patterns, were consistent with members of the genus Bartonella. Based on these results, we conclude that these three isolates are members of a novel species of the genus Bartonella for which we propose the name Bartonella ancashensis sp. nov. (type strain 20.00T = ATCC BAA-2694T = DSM 29364T).