Zooplankton transfer ecologically important fatty acids (FA) from their diets to upper trophic levels. We used diet-switching experiments with 13C-labeled food sources to determine the time scale at which dietary uptake is manifested in the FA profiles of Daphnia magna. Daphnia dramatically shifted their FA composition in response to diet change within only four days, however Daphnia switched from a high quality (i.e. Cryptomonas) to a moderate quality (Scenedesmus) diet retained the most physiologically important FA from their original diet source even after 14 days. In particular, Daphnia exhibited long-term retention of eicosapentaenoic (EPA; 20:5ω3) and arachidonic acid (ARA; 20:4ω6) when switched from Cryptomonas to Scenedesmus. Similarly, when switched from Scenedesmus to Cryptomonas, Daphnia took up a high proportion of EPA and ARA after only two days. The phospholipid fatty acid (PLFA) fraction in Daphnia was preferentially enriched with stearic (18:0), oleic (18:1ω9), and linoleic acid (LIN; 18:2ω6). In contrast with studies of marine copepods, dietary FA also strongly affected the PLFA composition (structural lipids) of Daphnia. Results of δ13C signatures of individual FA provided evidence of elongation and desaturation of α-linolenic (ALA; 18:3ω3) or stearidonic acid (SDA; 18:4ω3) to EPA 10 days after a diet switch to EPA-deficient Scenedesmus. Differences in the ARA content of Daphnia fed Cryptomonas and Scenedesmus suggest Daphnia consuming Cryptomonas synthesized ARA via retroconversion of ω6-docosapentaenoic acid (ω6-DPA; 22:5ω6). Daphnia preferentially accumulate and retain, as well as bioconvert, those FA that are also most physiologically important for fish production. Our results also indicate Daphnia FA composition responds to their diet on a short temporal scale and analyses of lipid biomarkers in zooplankton provide strong insights into the food sources that support their production.