Demodex mites are common commensals of mammalian skin, but under certain conditions, they can cause severe skin diseases. This study analyzed the presence, diversity, and phylogenetic relationships of Demodex species in two wolf subspecies from southern Europe to determine whether species-level differences exist between wild and domestic canids after thousands of years of divergence. A total of 1,400 hair samples from 140 wolves were analyzed using qPCR targeting mitochondrial 16S rRNA and nuclear 18S rRNA genes. Overall, 37% of wolves were positive for Demodex DNA, with a higher prevalence in Italian (46%) than in Iberian (36%) wolves. The lip and chin areas were the most reliable sampling sites. Four Demodex species were identified in wolves: D. injai and D. canis (associated with dogs), and D. folliculorum and D. brevis (associated with humans). Co-infestations involving multiple Demodex species were recorded for the first time in wild canids. These results challenge the long-held belief of strict host specificity in Demodex mites. The discovery of Demodex species associated with both humans and dogs in wolves supports the idea that host-switching and ecological interactions have occurred throughout the evolution of canids and humans. Such cross-species transfers may have taken place during the early domestication of dogs. However, given the isolated history of the two southern wolf populations, it is more probable that these findings result from recent interspecific transmission events, likely facilitated by ecological overlap with domestic animals and human environments. Future genomic studies will be essential for clarifying the evolutionary relationships within the genus Demodex and its host associations.