During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust markers to monitor it in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total lipid extracts of heads, female and male genital tracts of adult flies. We found that their sterol composition is dynamic and reflective of fly diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior, including cold acclimation. It provides technically simpler and more specific readout compared to the full lipidome analysis and is suitable for ecological and environmental population-based studies.