Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended.