There is evidence for the ecological extinction of the native prey of the puma Puma concolor in north-western Argentine Patagonia. In this study we examine whether this is also the case in southern Patagonia. From 2004 to 2007 we examined the puma’s diet in three protected areas and two sheep ranches in Santa Cruz province. A total of 282 puma scats were analysed. In two of the protected areas and in the ranches 60–74% of the puma’s diet was native prey. Prey species were primarily guanaco Lama guanicoe, followed by Patagonian mara Dolichotis patagonum, lesser rhea Pterocnemia pennata pennata, Patagonian pichi Zaedyus pichiy and Magellanic penguin Spheniscus magellanicus. In the third protected area the main prey was the European hare Lepus europaeus. Our results show a clear difference in the diet of the puma in southern compared to north-western Patagonia. Large native herbivores (i.e. guanaco and lesser rhea) maintain their role as the main prey species for the puma in southern Patagonia. We suggest, therefore, that native prey could be restored to those areas of Argentine Patagonia, such as the north-west, where they are currently ecologically extinct. Facilitating native species recovery and/or restoration and applying more rigorous controls to prevent the introduction of potential alien prey species of the puma both, within and outside protected areas, needs to be evaluated as a regional strategy.