Fatty acids composition of two marine copepods, Acartia tonsa and Centropages brachiatus, and lipid profiles of natural food assemblages were studied during the austral summer 2006 at three upwelling sites in the coastal upwelling zone off Chile, along with oceanographic conditions. Fatty acids of food supply were assessed for the picoplankton, nanoplankton and microplankton size fractions. There were marked differences in upwelling conditions among locations, as well as in their food supply in terms of quantity and quality. Differences in fatty acid composition were also found, both among food assemblages and between copepod species. Essential polyunsaturated fatty acids (PUFA; linoleic acid) and monounsaturated fatty acids (MUFA; oleic acid) dominated the picoplankton and nanoplankton size fractions of food, and they were highly represented in both species of copepods, indicating these size fractions were the major contributors to their diet. These fatty acids can thus be considered as useful trophic markers for copepods. Variation in lipid profiles between species depended on sampling sites, whereas differences in lipid composition among sampling sites were attributed to distinct upwelling conditions, which drive the changes in food quality, such that trophic response is highly dependent on food offer. Variation in fatty acids compositions of copepods may thus act as an indicator of upwelling variability. Our findings suggest that lipid transfer from primary producers to primary consumers can have a crucial role for carbon cycling in the marine food web, and that picoplankton and nanoplankton fractions are the key items of copepod diet in this upwelling system.