Dietary habits of free-ranging animals are essential for understanding their ecology, conservation and management. Carnivore diet is most frequently estimated using morphological analysis of prey remains found in scats. However, genetic methods may identify prey in scats when parts are too small to identify by morphological methods. We developed an easy and accurate molecular approach to assess occurrence of prey species in the diet of free-ranging wolves (Canis lupus) and compared the results to analyses of prey hair in the same scat samples collected from northeastern Alberta, Canada. Species-specific mitochondrial DNA primers detected 1.34 times more prey occurrences from scats, and more readily differentiated similar ungulate species such as woodland caribou (Rangifer tarandus caribou) and deer (Odocoileus sp.) than morphological methods. These findings suggest that molecular analysis of prey in carnivore scat are objective, reproducible and, can help promote effective conservation and management of carnivore species at risk of conflict with humans.