Human food waste is an essential resource for many animals that inhabit urban ecosystems and has allowed some species to proliferate in cities. Virginia Opossums ( Didelphis virginiana ) are considered a good example of this process; however, the real role of waste in this animal’s diet is still unclear. Therefore, this study aims to compare opossums’ diet from urban and natural areas in Mexico City. Opossums were live-trapped, and samples of hair and blood were taken to perform a stable isotope analysis of C and N. The results show that opossums from natural and urban areas, in general, have a very similar isotopic niche; however, one natural location fell out of this pattern, and here opossums based their diet on C3 plants. This suggests that opossum diets have a similar carbon source and occupy a similar trophic position in urban and natural areas. However, there is a separation between opossums in urban and natural areas, not so much in the isotopic niche, but in the feeding habits since natural areas, opossums prefer to feed in their distribution area despite the availability of better quality food in the nearby urban areas.