We conducted a series of experiments feeding Daphnia pulex nine different phytoplankton monocultures with widely varying fatty acid composition and nutritional values to test the extent to which Daphnia fatty acid composition was affected by diet. In general, Daphnia fatty acid composition matched that of their diet much more closely than it did the fatty acid composition of Daphnia consuming other diets. However, Daphnia had consistently less saturated fatty acids and more arachidonic acid than did their diet, and Daphnia consuming cyanobacteria had substantially less saturated fatty acids and more monounsaturated fatty acids than their diets. Daphnia that consumed cryptophytes, which are rich in ω3 polyunsaturated fatty acids (PUFAs), had on average 47% ± 8% (±1 SD) ω3 PUFAs within their fatty acid pool, whereas Daphnia that consumed ω3 PUFA-poor cyanophytes only had 6% ± 3% ω3 PUFAs. The ratio of ω3 to ω6 fatty acids in Daphnia was also strongly dependent on diet, and averaged ≈ 10:1, 2:1, and 1:1 for Daphnia that consumed cryptophytes, chlorophytes, and cyanophytes, respectively. Furthermore, the sum of C20 and C22 ω3 and ω6 fatty acids in Daphnia was highly correlated with that of their diet (r2 = 0.94). These results suggest analyses of Daphnia fatty acid composition may be a powerful means of inferring diet in the field. These results also suggest the nutritional benefits of consuming ω3-rich phytoplankton will transfer up the food web, making zooplankton both more efficient at converting phytoplankton biomass to their own biomass as well as much more nutritious for the zooplanktivorous fish that consume them.