During two vegetation seasons (2004–2005), we compared feeding spectra of Arctodiaptomus salinus (Calanoida, Copepoda) populations inhabiting two neighboring salt lakes, Shira and Shunet, Khakasia, Russia, using fatty acid (FA) trophic markers. Sestonic FA composition in two lakes moderately differed, whereas levels of diatom FA markers were higher in Lake Shunet and of Cyanobacteria and green algae markers in Lake Shira. In general, markers in storage lipids—triacylglycerols (TAG) of A. salinus—reflected the differences in sestonic composition of the two lakes. Nevertheless, TAG fraction was also enriched by FA trophic markers of the minor components of seston, which were selectively ingested by the animals. In Lake Shira, A. salinus had significantly higher concentrations of bacterial FA markers in TAG. In Lake Shunet, TAG of A. salinus contained significantly higher relative amounts of 18:4ω3, 18:5ω3 and C22 polyunsaturated fatty acids (PUFA), which indicated marked contribution of cryptophytes or (and) flagellates into the diet. Laboratory experiments showed feeding on Cryptomonas and sulfur purple bacteria in Lake Shunet and ciliates and colonial picoplankton in both lakes, and generally confirmed the differences in FA trophic markers in A. salinus between the lakes. The two populations of A. salinus markedly differed in levels of essential long-chain PUFA, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, although the levels of these FA in seston were quite similar between the two lakes. The higher levels of the essential ω3 PUFA in A. salinus in Lake Shunet may be an adaptive response of the animals to a vertical stratification of physico-chemical conditions and significantly higher salinity levels at the boundary of adjacent bottom layer in this lake.