As climate change, among other factors, is increasingly affecting Antarctic marine systems, competition for prey may increase between predators, particularly in the Antarctic Peninsula which has warmed more than elsewhere. Under such a context, we tested the feeding and trophic ecology of Gentoo (Pygoscelis papua) and Chinstrap (Pygoscelis antarctica) penguins breeding in sympatry at Livingston Island (Antarctic Peninsula) in a single season. We compared the diets of adults (from faecal samples, and stable isotopes in feathers and blood) and chicks (from stomach contents, and stable isotopes in down feathers, toenails and muscles of chicks that had died of unknown causes). Antarctic krill Euphausia superba dominated the diet of both species, although Gentoo Penguins fed on larger Antarctic krill than did Chinstrap Penguins. Stable isotope analyses of adult tissues revealed that both species fed at different niches in successive years, as depicted by the different levels δ13C in feathers (showing values from the previous breeding season) and whole blood (showing values from the current season). Tissues collected from chicks confirmed their diet over different time scales (i.e. days to weeks): Gentoo Penguins fed at a higher trophic level (possibly due to a more varied diet) and in different habitats than Chinstrap Penguins, providing evidence of isotopic niche separation of penguins. Our results may be relevant to the monitoring programmes of the Convention on the Conservation of Antarctic Marine Living Resources (CCAMLR) and suggest that adult scats, and stomach contents and tissues of recently died chicks, can be used in such programmes.