Visceral Leishmaniasis (VL) is an emerging zoonotic parasitic disease caused by Leishmania infantum in Mediterranean countries, with sand flies (Phlebotomus spp.) as vectors and dogs as the main domestic reservoir. The role of wild carnivores in the epidemiology of leishmaniasis is still controversial. In order to determine the prevalence of natural infection with L. infantum in wild carnivores from Spain, we analyzed 217 samples by PCR and western blotting and used restriction fragment length polymorphism (RFLP) to compare the patterns present in wild carnivores with those of domestic dogs from the same areas. DNA of the parasite was detected in spleen or blood samples from 35 (16.12%) analyzed wild carnivores, including 8 of 39 (20.5%) wolves (Canis lupus), 23 of 162 (14.1%) foxes (Vulpes vulpes), 2 of 7 (28.6%) Egyptian mongooses (Herpestes ichneumon), 1 of 4 genets (Geneta geneta), and 1 of 4 Iberian lynxes (Lynx pardinus). No significant sex or age differences in prevalence were observed in wolves and foxes (P>0.05), but there was a significant difference among regions in foxes (P<0.05). A total of 12 PCR-RFLP patterns were found in foxes, 6 in wolves, 4 in dogs, 2 in Egyptian mongooses and 1 in lynx and genet. RFLP patterns differed between dogs and foxes in the two areas where they could be compared. This is the first study of canine leishmaniasis in wild canids and other carnivores from different regions of Spain by PCR. The prevalence of infection indicates the existence of natural infection in apparently healthy wild carnivore populations, and our results are suggestive of a sylvatic cycle independent of dogs.