This paper studies the fatty acid (FA) composition and content of essential polyunsaturated fatty acids (PUFAs) in the biomass of larvae and adults of chironomids from the saline Shira Lake. Species of different genera significantly differ in their larvae FA composition and essential PUFA content, and they also occupy different ecological niches: Chironomus species with a low PUFA content (0.2–0.3 mg g–1 of wet weight) inhabit a deepwater zone of the lake, while Glyptotendipes barbipes species that were richer in PUFA (2.3 mg g–1 of wet weight) dwell in the littoral of the lake. The biochemical differences are likely related to different feeding spectra of these taxa and can also be explained by the phylogenetic factor. A comparison does not find differences in the PUFA content in larvae and adults in the samples of the same species G. barbipes; i.e., we do not confirm the data on an increase in the content of these acids during the metamorphosis of chironomids. Thus, the data on the PUFA content in larvae can be used in calculations of PUFA fluxes through chironomid emergence from water bodies; however, the taxonomic affiliation of the emerged chironomids should be taken into consideration due to the high variability in the PUFA content in Chironomidae species.