Fatty acids are crucial for energy transfer within food webs, acting as biomarkers due to their persistence across trophic levels. The anchoveta is economically and ecologically important, with extensive research on its biology. However, the impact of diet on its fatty acid composition across different regions and sizes is less understood. Fatty acids were extracted, saponified, and prepared as fatty acid methyl esters for analysis on a gas chromatograph. This study reveals that anchoveta have high levels of saturated fatty acids (SAFAs) and primarily use docosahexaenoic acid (DHA) as their energy source. We also found that the fatty acid profile of anchoveta changes throughout their ontogeny, with prerecruits having lower DHA levels compared to adults. Interestingly, anchoveta from similar geographic regions like Chile, Peru, and South Africa share similar fatty acid profiles. This suggests a potential link to shared upwelling habitats that are rich in nutrients. Furthermore, our study challenges the traditional assumption that phytoplankton is the main food source for anchoveta. The results highlight the need for further research to pinpoint their primary energy source.