Babesia spp. are globally distributed tick-borne protozoan parasites that infect the red blood cells of a wide range of vertebrate hosts, including humans. Diagnosis of babesiosis is often impeded by the transient presence of the parasites in peripheral blood, as well as by their pleomorphic nature. Given the reports of an expanding and, in some cases, sympatric geographical distribution of Babesia canis and Babesia vogeli in dogs and associated vectors, in Europe, the development of time-efficient and cost-effective diagnostic tools to detect and differentiate these two species is warranted. In this study, we designed and developed a novel polymerase chain reaction (PCR) assay targeting the parasite cytochrome c oxidase subunit 1 (cox1) gene, for the simultaneous detection and differentiation of B. canis and B. vogeli. The analytical sensitivity of the PCR was evaluated using serial dilutions of genomic DNA extracted from individual and artificially mixed canine blood samples infected by B. canis (3×102 infected erythrocytes/ml, ie/ml) and B. vogeli (2.1×101 ie/ml). The analytical specificity of the assay was assessed using blood samples positive for Hepatozoon canis, Ehrlichia canis, Anaplasma platys, Babesia microti, Babesia rossi and Theileria annae (syn. Babesia vulpes). The clinical specificity of the PCR assay was evaluated on 147 blood samples from dogs and 128 tick specimens (Dermacentor reticulatus and Rhipicephalus sanguineus sensu lato). Species-specific bands of the expected sizes (i.e., 750bp for B. canis and 450bp for B. vogeli), and two bands in the mixed blood samples were obtained. The PCR assay developed herein detected a low number of infected erythrocytes (i.e., 3×10-2B. canis, 2.1×10-2B. vogeli ie/ml). Of the 147 blood samples, nine (6.1%) were positive for B. canis and six (4.1%) for B. vogeli, whereas only one tick (D. reticulatus) was positive for B. canis. This PCR assay represents a rapid and reliable tool for the diagnosis of B. canis and B. vogeli in areas where these species occur in sympatry, and will assist future epidemiological investigations aimed to track the expanding range of these parasites.