Canine babesiosis (or piroplasmosis) is an emerging tick-borne disease caused by intraerythrocytic protozoa of the genera Babesia and Theileria. In dogs, infection by these parasites usually induces a haemolytic syndrome that can be fatal when complicated. Canine babesiosis prevalence is high in France, with Babesia canis thought to be the main etiological agent of the disease. This article presents the results of a multiregional prospective longitudinal survey on canine babesiosis conducted in France from October 2006 to December 2007. A total of 836 cases were reported by veterinarians using a multiple choice questionnaire and blood samples from 70 dogs were analyzed using PCR-RFLP to identify species responsible for canine babesiosis cases across the country. The main clinical signs reported were lethargy (98%), anorexia (98%) and hyperthermia≥39°C (80%) followed by pale mucous membranes (54%), modification of urine aspect (45%) and splenomegaly (33%). The dog population at risk was mainly represented by young dogs living in rural areas. Twenty-five out of the 70 blood samples (36%) tested by diagnostic PCR were found to contain Babesia/Theileria genus-specific DNA and all had profiles similar to that of Babesia canis genomic DNA after restriction fragment length polymorphism analyses. The survey results provide a reference for further molecular studies to assess the species and vectors involved in the transmission of the disease in France and across the Mediterranean basin.