The present study aimed to determine the tissue-specific robustness of fatty acid (FA) signatures of gilthead sea bream (Sparus aurata L.) by analyzing the changes in lipid class and FA composition of skeletal muscle, brain, liver, and mesenteric adipose tissue. Triplicate groups of fish were fed to visual satiety over a 14-mo production cycle with 2 practical plant protein-based diets formulated with either fish oil or a blend of vegetable oils (66% of fish oil replacement) to contain 53% CP and 21% crude fat on a DM basis. Growth rates (P = 0.22) and tissue lipid class composition were not altered by the dietary treatment (P = 0.34 and 0.52 for neutral lipids and phospholipids, respectively). The FA signatures of neutral lipids reflected the composition of the diet, although the output of principal components analysis revealed a divergent FA profile for liver compared with skeletal muscle, brain, and mesenteric adipose tissue. Because the theoretical EFA needs were met by the 2 diets, the FA composition of phospholipids remained almost unaltered in all tissues. Interestingly, however, the brain showed the greatest robustness and regulatory capacity to preserve the phenotype of fish fed fish oil-based diets. The FA signatures of total lipids are a combinatory result of neutral and polar lipids, and the most relevant fat storage tissues (mesenteric adipose tissue and skeletal muscle) were more easily influenced by dietary FA composition. The present study provides new insights into fish tissue FA composition and reinforces the use of FA signatures as useful criteria in determining whether EFA requirements for a wide range of physiological processes, including those of neural tissues, can be met with practical fish feeds.