Yellowfin tuna (Thunnus albacares) is an apex predator in marine ecosystems and plays a vital role in energy flow and nutrient cycling. This study aimed to investigate the dietary modifications of Pacific yellowfin tuna throughout ontogeny and variations in nutritional niches among different maritime regions. The fatty acid composition and properties of yellowfin tuna muscle were examined using specimens obtained from tuna fishing vessels operating in the eastern Pacific Ocean in 2019 and the western and central Pacific Ocean in 2021. Thirty-four fatty acids in yellowfin tuna muscle, including 16 saturated fatty acids (SFA), 8 monounsaturated fatty acids (MUFA), and 10 polyunsaturated fatty acids (PUFA), were identified. The SFA content of yellowfin tuna muscle was negatively correlated with the PUFA content, whereas the MUFA content was positively correlated with the PUFA content. Furthermore, a significant negative correlation was observed between SFA content and fork length, whereas both MUFA and PUFA content exhibited a significant positive correlation with fork length. Distinct variations were observed in the muscle fatty acid composition of yellowfin tuna inhabiting the western and central Pacific Oceans compared to those in the eastern Pacific Ocean, with an average compositional dissimilarity of 18.85%. Primary disparities were found in n-3 polyunsaturated fatty acids (n-3 PUFA), SFA, PUFA, C22∶6n3 (DHA), Cl6∶0, Cl8∶1n9, MUFA, C22∶6n3/C20:5n3 (DHA/EPA), C18∶0, and n-6 series of polyunsaturated fatty acids (n-6 PUFA). Principal component analysis showed that the higher the trophic level of yellowfin tuna, the higher the carnivorous ability of yellowfin tuna and the greater the contribution of organisms that feed on dinoflagellates and diatoms to the energy sources of yellowfin tuna. In contrast, yellow-fin tuna at lower trophic levels mainly feed on bacteria, terrestrial plants, and phytophagous copepods. Principal component regression analysis indicated that, as fork length increased, the nutritional level and carnivorous nature of yellowfin tuna also increased. Additionally, their feeding behavior progressively shifts from relying on crustaceans and cephalopods to organisms abundant in characteristic fatty acids, such as shrimp and teleosts. Compared to the yellowfin tuna inhabiting the western and central Pacific Oceans, those in the eastern Pacific Ocean exhibit a higher degree of carnivorous behavior and occupy higher trophic levels, with crustaceans contributing significantly to their energy intake.