Food habits of cougars (Puma concolor) in North America have been documented for western populations in the United States, Canada and Mexico. Most studies assessed diets of cougars occupying typical habitats, and within established populations. We evaluated food habits of cougars in prairie and agricultural landscapes in the Dakotas (regions that had been devoid of the species for roughly a century) located well outside of known resident populations. We obtained stomach and gastrointestinal (GI) tracts from 14 cougars (10 male; 4 female) from 2003–2007, and evaluated contents via frequency of occurrence (%) of various prey items. Deer (Odocoileus spp.) had the highest frequency of occurrence (50.0%). Other native mammalian prey included jackrabbit (Lepus townsendii, L. californicus), porcupine (Erethizon dorsatum), beaver (Castor canadensis), badger (Taxidea taxus), mink (Mustela vison) and rodent species (e.g., vole). No domestic livestock species were documented as part of the cougar diet in the Dakotas, although remains of domestic housecat (Felis silvestris) were found in GI tracts of two animals. Based on our results, cougars occupying non-typical, newly recolonized habitats were successfully adapting predation techniques for capture of natural and newly confronted prey species. The wide range of prey encountered suggested that prey was being obtained opportunistically in prairie and agricultural landscapes of the Dakotas.