Fatty acid (FA) content and composition of zooplankton in Puget Sound, Washington (USA) was studied to investigate the nutritional quality of diverse zooplankton prey for juvenile salmon (Oncorhynchus spp.) in terms of their essential fatty acid (EFA) content. The study focus was on eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA) as these are key FA needed to maintain growth and development of juvenile fish. The different zooplankton taxa varied in their FA composition. Much of the variation in FA composition was driven by 18:1ω9 (a biomarker of carnivory), ARA, DHA, and FA characteristic of diatoms, which are linked to zooplankton diet sources. Gammarid and hyperiid amphipods contained the highest amount of EFA, particularly the gammarid amphipod Cyphocaris challengeri, while shrimp and copepods had much lower EFA content. Crab larvae, which are important prey for juvenile salmon in Puget Sound, had intermediate EPA + DHA content and the lowest DHA/EPA ratio, and were rich in diatom biomarkers. Temporal and spatial trends in zooplankton lipids were less apparent than the taxonomic differences, although the EFA content increased from spring to summer in Cancridae zoeae and the amphipod C. challengeri. These results on taxon-specific EFA content provide baseline information on the nutritional quality of zooplankton that can be applied in food web models. Combining zooplankton fatty acid data (quality) with taxon-specific zooplankton biomass data (quantity) enables development of new, sensitive indicators of juvenile fish production to help assess recent declines in salmon production in the Pacific Northwest and predict future adult returns.