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A previously established gas chromatography-mass spectrometry method was applied for the determination of the fatty acid composition in the muscles of Atlantic bluefin tuna (Thunnus thynnus L). The fish were collected in the Mediterranean Sea and cultivated for several months. The goal of the study was to demonstrate the feasibility of these measurements and to improve knowledge about the influence of size and gender on the fatty acid composition. Although no significant differences were found with respect to gender, males and females showed different correlations of fatty acid composition with weight. In the males, a statistically significant linear correlation was found for 14:0, 16:0, 18:1n9, 20:1n9, 22:1n9 (positive correlation), and for 20:4n6, 20:5n3 22:6n3 (negative correlation). In the females, a statistically significant polynomial correlation for specific fatty acids was highlighted: specimens with medium size showed the lowest content of 20:1n9 and 22:1n9 and the highest content of 20:4n6, 20:5n3, and 22:6n3. Therefore, males smaller than 150 kg and females from 150 to 250 kg appeared to have the highest greater nutritional value. This study demonstrated the practicality of the analytical methodology and enhanced the knowledge of the influence of size on the fatty acid composition of Atlantic bluefin tuna muscle, reporting a newly established gender difference and representing a starting point to produce the best nutritional characteristics for farmed tuna.