Fatty acids are relatively stable in the feeding activities of organisms and can be used to identify consumers' food sources. The carbon source composition of zooplankton in lakes and reservoirs with different nutrient levels is different, so they may have different fatty acid composition characteristics. This study compared the fatty acid composition of POM (particulate organic matter) and zooplankton in 21 lakes and reservoirs in Nanjing and its surrounding areas. The results show that the 21 lakes and reservoirs are composed of four nutritional levels:medium nutrition, light eutrophication, medium eutrophication, and heavy eutrophication. The total nitrogen (TN), total phosphorus (TP), chlorophyll-a (Chl.a), and phytoplankton biomass of mesotrophic lakes and reservoirs are lower than those of eutrophic lakes and reservoirs, while the transparency (SD) is the opposite. The average concentration of essential fatty acids (EFA) of POM fatty acids in mesotrophic lakes was lower than that of eutrophic lakes, while the content of bacterial characteristic fatty acids ∑C15+∑C17 was higher than that of eutrophic lakes. With nutrient increase, the concentration of polyunsaturated fatty acids (PUFA) in POM gradually increases, while the concentration of monounsaturated fatty acids (MUFA) gradually decreases. The PUFA of zooplankton in mesotrophic lakes and reservoirs is generally lower than that of eutrophic lakes and reservoirs, and the content of ∑C15+∑C17 is higher than that of eutrophic lakes and reservoirs; and the ratio of essential fatty acids EFA/C16:0, which are mainly derived from algae, and bacterial fatty acids in zooplankton (The ratio of ∑C15+∑C17)/C16:0 is larger than that of other lakes and reservoirs, indicating that zooplankton in the middle-nutrition lakes and reservoirs ingested more bacteria. As nutrient increases, zooplankton saturated fatty acids (SFA) gradually increase, MUFA gradually decreases, and EFA content increases, indicating that zooplankton mainly feeds on algae, while exogenous carbon contributes little to the carbon source of zooplankton. Redundancy analysis (RDA) show that EFA/C16:0, (∑C15+∑C17)/C16:0 are positively correlated with SD, and highly negatively correlated with Chl.a, TP, and phosphate, suggesting that zooplankton consumed more algal and bacterial carbon; and ∑C24/C16:0 is positively correlated with TN, nitrate nitrogen, indicating that zooplankton utilized more terrestrial carbon. In general, zooplankton is more inclined to use high-quality endogenous carbon, but in mesotrophic water bodies where endogenous carbon is not sufficient to supply the growing needs of aquatic organisms. Exogenous carbon plays an important role in the material circulation and energy flow of the ecosystem.