Knowledge of the dietary choices and trophic niches of organisms is the key to understanding their roles in ecosystems. In seabird diet studies, prey identification is a difficult challenge, often yielding results with technique-specific biases. Additionally, sampling efforts are often not extensive enough to reveal intra-populational variation. Immature animals, which may constitute up to 50% of a population, may occupy a significantly different trophic niche to more-experienced birds, but this remains largely unexplored. We investigated the diet of Cory′s shearwater (Calonectris diomedea) from Selvagem Grande, an island located off the northwest African coast, collecting a total of 698 regurgitate samples over three consecutive breeding seasons. The diet was assessed using two complementary approaches for prey identification: conventional morphological analysis (using fish vertebrae, otoliths and cephalopod beaks) and DNA barcoding of the 16S rRNA mitochondrial gene, in cases where a positive identification could not be retrieved. Species assignments employed BLAST and distance based methods, as well as direct optimization of the tree length based on unaligned sequences in POY. This method resulted in robust tree estimates and species assignments, showing its potential for DNA barcoding of stomach contents using hypervariable markers such as the 16S. The molecular approach increased taxonomic resolution and revealed an additional 17 taxa. Diet differed significantly according to breeding status, sex, breeding phase (pre-laying and chick-rearing) and year. Such direct evidence of trophic segregation within the same population has rarely been shown in seabirds and highlights the importance of including such variables in ecosystem-based management approaches.