Some procellariiform seabirds use a dual strategy for provisioning their chicks by alternating between short and long foraging trips (LT). Trophic relationships of adult birds are unknown when they feed for themselves during LT because digestion processes preclude direct prey determination. Since stomach contents collected after LT contain oil of dietary origin, we tested the use of oil lipids as prey trophic markers using the Tasmanian short-tailed shearwater Puffinus tenuirostris as a model seabird. The intra-specific variability of stomach oils was investigated through lipid class composition, and their fatty acid and fatty alcohol profiles. Oils mainly consisted of wax esters (WE) and triacylglycerols (TAG) (49 to 86 and 7 to 41%, respectively). Major fatty acids of TAG were in a decreasing order 18:1n-9, 16:0, 16:1n-7, 14:0, 20:5n-3 and 22:6n-3. The WE-fatty acid profiles were dominated by 18:1n-9 and 16:1n-7 while fatty alcohol profiles were dominated by 16:0. Fatty alcohol and fatty acid patterns were tested as possible descriptors of ingested prey (derived from literature data) through multivariate discriminant analyses. Comparisons of the WE fatty alcohol patterns showed a close association with the alcohol structure of 3 myctophid fish species namely Krefftichthys anderssoni, Gymnoscopelus braueri and Electrona antarctica; these results were corroborated by WE fatty acid analysis. Comparison of TAG fatty acid patterns showed the highest similarity between oils and the digestive gland of the myctophid-eater squid Moroteuthis ingens in association with the myctophid Electrona carlsbergi. Hence, biochemical analysis of both WE and TAG strongly suggested that adult short-tailed shearwaters mainly prey upon Antarctic/sub- Antarctic myctophids when they feed for themselves, thus emphasizing the role of these oceanic mesopelagic fish in the marine ecosystem of the Southern Ocean.