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A comprehensive lipidomic analysis of the lipids extracted from grass carp bones, black carp bones, shrimp shells, and crab shells was performed in this study. First, HPLC analysis revealed that the lipids extracted from shrimp and crab shells contained 60.65% and 77.25% of diacylglycerols, respectively. Second, GC‐MS analysis identified 18 fatty acid species in the lipids extracted from fish bones and crustacean shells, in which polyunsaturated fatty acids (PUFAs) were highly enriched. PUFAs were present at 45.43% in the lipids extracted from shrimp shells. Notably, the lipids extracted from shrimp and crab shells contained a considerable amount of eicosapentaenoic acids and docosahexaenoic acids. Finally, multidimensional mass spectrometry‐based shotgun lipidomics showed that various lipids including acetyl‐L‐carnitine, sphingomyelin (SM), lysophosphatidylcholine, and phosphatidylcholine (PC) were all identified in the lipid samples, but PC and SM were the most abundant. Specifically, the total content of PC in shrimp shells was as high as 6.145 mmol/g. More than 35 species of PC were found in all samples, which were more than other lipids. This study is expected to provide a scientific basis for the application of freshwater fish bones and crustacean shells in food, medicine, and other fields. [ABSTRACT FROM AUTHOR]