The zooplankton community is a critical link between primary producers and higher trophic organisms including fish and birds and they are often the main conduit of energy through aquatic food webs. Zooplankton require a supply of specific long chain polyunsaturated fatty acids, or essential fatty acids (EFA), in their diet to ensure growth and reproduction. There are multiple sources of these essential fatty acids in lake and marine systems. We conducted laboratory feeding experiments with two distinct types of important zooplankton, daphnids and calanoid copepods, to evaluate the use of compound specific stable isotope analysis of specific fatty acids as a means for tracing the movement of these essential compounds between an organism and its diet. Most fatty acids in daphnids had similar stable carbon isotope values as those of their algal diet, indicating the potential to trace the movement of fatty acids between a consumer and its diet. In contrast, the turnover of most fatty acids in the copepods was slower, however the stable carbon isotope composition of the EFA 18:3w3 in the copepods did reflect the isotopic signature of this EFA in their dietary algal source. We suggest that the stable isotope composition of 18:3w3 can be used to trace the flux of EFA through aquatic food webs. This compound specific stable carbon isotope approach was verified in three lake systems through similar analyses of algae, Daphnia, and Bythotrephes over several months. These results indicated that zooplankton obtained their EFA from only a portion of the algal community.