Here, we describe a partial cranium of a large canid dated at 406.5 ± 2.4 ka from the Middle Pleistocene of Ponte Galeria (Rome, Italy). The sample represents one of the few Middle Pleistocene remains of a wolf-like canid falling within the timeframe when the Canis mosbachensis-Canis lupus transition occurred, a key moment to understand the spread of the extant wolf (Canis lupus) in Europe. CT-based methods allow studying the outer and inner cranial anatomy (brain and frontal sinuses) of a selected sample of fossil and extant canids. Morphological and biometric results allowed to: (I) ascribe the cranium from Ponte Galeria to an adult Canis lupus, representing the first reliable occurrence of this taxon in Europe; (II) provide the content for a biochronological revision of the Middle Pleistocene record of European wolves.