We determined the δ15N and δ13C values of individual amino acids (AAs) isolated from chick blood of 4 penguin species that forage in different oceanic regions (from the subtropics of the Indian Ocean to Antarctica) to test if: (1) the δ15N values of phenylalanine (δ15N phe) revealed different foraging areas among the species; (2) the difference between glutamic acid and phenylalanine δ15N values (Δδ15Nglu-phe) accurately predicted trophic levels; and (3) the δ13C value of AAs could resolve species foraging locations, similar to bulk δ13C values. The δ13C values of all AAs decreased with latitude, were positively correlated with bulk δ13C data, and, therefore, tracked the isotopic baseline. However, we were not able to discern additional ecological information from these δ13C values. In contrast, the δ15N values of AAs distinguished the isotopic value of the nitrogen at the base of the food web from the trophic level of the consumer, providing new insight for the study of the trophic ecology of seabirds. The difference in the bulk δ15N values of northern and southern rockhopper penguins Eudyptes chrysocome ssp. was due to both a difference in their foraging location (different δ15N phe) and their trophic levels (different Δδ15Nglu-phe). The δ15N phe values of king Aptenodytes patagonicus and Adélie penguins Pygoscelis adeliae were higher than those of rockhoppers, which could reflect a foraging on mesopelagic prey for king penguins and, in the highly productive Antarctic shelf waters, for Adelie penguins. The Δδ15Nglu-phe accurately reflected the relative trophic level of penguins, but further work is required to determine the trophic enrichment factors for compound-specific isotope analysis.