This is the first report on the presence of Hepatozoon canis in Vulpes vulpes in Italy. During the years 2005 and 2006, a total of 119 foxes were collected and their spleen tissues were screened by microscopy, polymerase chain reaction, and sequencing. In the same area, 290 ticks were picked off from dogs or collected from the environment. Microscopy detected inclusion bodies regarded as belonging to the genus Hepatozoon in four samples, whereas molecular diagnostics evidenced 16 foxes (13.4%) and 6 ticks (2.1%) positive to H. canis. The H. canis isolates we found in foxes, compared with the strains we previously detected in dogs from the same area and with the strains found in foxes from other European countries, show a certain genetic heterogeneity. In fact, seven isolates cluster with the Italian dog strain and nine isolates cluster with the fox strain found in Spain and Slovakia; moreover, the dog's strain is closely related to one tick's isolate, and the strain found in three Rhipicephalus sanguineus and in one Ixodes ricinus collected from the environment cluster with the aforementioned Spanish and Slovak fox strains. Our findings confirm the importance of R. sanguineus as final host and suggest that I. ricinus might also be implicated in parasite transmission, explaining in that way the occurrence of hepatozoonosis in areas considered R. sanguineus-free. The peridomestic habits of V. vulpes and the increasing global temperature are expected to amplify the impact of this vector-borne disease and to enforce the transmission of Hepatozoon to domestic animals.