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The variability in zooplankton fatty acid composition may be an indicator of larval fish habitat quality as fatty acids are linked to fish larval growth and survival. We sampled an anadromous fish nursery, the Chowan River, during spring of 2013 in order to determine how the seston fatty acid composition varied in comparison with thezooplanktoncommunitycompositionandfattyacidcompositionduringtheperiod of anadromous larval fish residency. The seston fatty acid profiles showed no distinct pattern in relation to sampling time or location. The mesozooplankton community composition varied spatially and the fatty acid profiles were typical of freshwater species in April. The Chowan River experienced a saltwater intrusion event during May,whichresultedinbrackishwaterspeciesdominatingthezooplanktoncommunity andthefattyacidprofileshowedanincreaseinpolyunsaturatedfattyacids(PUFA),in particulareicosapentaenoicacid(EPA)anddocosahexaenoicacid(DHA).Thesaltwater intrusioneventwasfollowedbyaninfluxoffreshwaterduetohighprecipitationlevels in June. The zooplankton community composition once again became dominated by freshwaterspeciesandthefattyacidprofilesshiftedtoreflectthischange;however,EPA levels remained high, particularly in the lower river. We found correlations between the seston, microzooplankton and mesozooplankton fatty acid compositions. Salinity was the main factor correlated to the observed pattern in species composition, and fatty acid changes in the mesozooplankton. These data suggest that anadromous fish nursery habitat likely experiences considerable spatial variability in fatty acid profiles of zooplankton prey and that are correlated to seston community composition and hydrodynamic changes. Our results also suggest that sufficient prey density as well as a diverse fatty acid composition is present in the Chowan River to support larval fish production.