Abstract: Many types of animal tissues are increasingly being used for stable isotope analysis, with their application dependent on the time frame they reflect and their availability for collection. Here, we investigated the isotopic values (δ13C and δ15N) of four tissues (feather, skin, toenail, and bone) collected from fledgling-period chick carcasses of three species of pygoscelid penguins to compare the variability and accuracy of the data among tissues. Samples were collected at 25 de Mayo/King George Island during the 2017/18 austral summer. Chick carcasses are commonly found at active penguin colonies, and “opportunistic sampling” can easily be performed without disturbing nesting penguins. A total of 25–36 carcasses per species were sampled at active colonies of Adélie Pygoscelis adeliae, Gentoo P. papua, and Chinstrap P. antarcticus penguins. A linear mixed model showed that δ13C values varied significantly between tissues, presumably due to tissue-specific isotopic discrimination. In contrast, the only tissue with significantly different δ15N values was toenail. Stable isotope data revealed dietary differences among species, with Gentoo Penguins having higher average isotopic values in tissues compared to Adélie and Chinstrap penguins. In addition, Chinstrap Penguins showed a consistent, but not statistically significant, trend in having higher δ13C values compared to Adélie Penguins. Gentoo Penguins displayed the highest isotopic variability of any species for all tissues. Isotopic composition was most variable in skin in all three species making skin the least reliable tissue for isotope analysis, whereas isotopic values were least variable in toenails. Comparison of isotopic values between two bones (tibiotarsus and coracoid) showed no significant differences in isotopic values, indicating that when the same bone is not available for sampling from carcasses, sampling of any major skeletal element is likely to provide a meaningful comparison. These results allow for more informed opportunistic sampling to accurately estimate and compare penguin diet among species and between ancient and active colonies.