Fatty acids are key biomarkers that act as essential energy substrates and structural components for aquatic organisms while displaying conserved, taxon-specific profiles across primary producer groups, making them effective tracers of nutritional sources and energy flow in aquatic ecosystems. Zooplankton, as critical links between primary producers and higher trophic levels, exhibit fatty acid compositions that reflect dietary quality and utilization efficiency, thereby governing the ecological efficiency of energy transfer to upper food webs. Lake Erhai, the second-largest plateau freshwater lake in Yunnan Province, China, has experienced increasing eutrophication and frequent cyanobacterial blooms in recent years, yet studies on zooplankton fatty acids in this lake remain limited. To examine seasonal variation in zooplankton fatty acid composition and its environmental drivers, four dominant species—Mesocyclops leuckarti, Daphnia galeata, Phyllodiaptomus tunguidus, and Bosmina longirostris—were collected monthly from July to October 2025 at five sampling sites in Lake Erhai. Fatty acids were analyzed by gas chromatography–mass spectrometry (GC–MS), and permutational multivariate analysis of variance (PERMANOVA), similarity percentage analysis (SIMPER), and redundancy analysis (RDA) were used to evaluate the effects of month, species, site, and environmental variables. Results indicated that: (1) month was the primary factor shaping fatty acid composition, accounting for 40.93% of variance (PERMANOVA, F=25.827, P<0.0001), with no significant main effects of species (R2=0.035, P=0.195) or site (R2=0.059, P=0.082); (2) phytoplankton community composition strongly influenced particulate organic matter (POM) fatty acid quality, with diatoms (especially Cyclotella) and Cryptomonas positively correlated with polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (ARA); (3) variance partitioning showed that POM fatty acids and environmental variables together explained 54.25% of zooplankton fatty acid variance, with pure effects of 28.43% (P=0.001) and 14.28% (P=0.006), respectively, and water temperature as the most influential single factor (R2=0.141, P=0.001); (4) SIMPER analysis revealed that declining water temperature significantly increased zooplankton polyunsaturated fatty acid (PUFA) abundance, with EPA rising from 3.78% in July to 7.86% in October and ARA from 3.41% in September to 9.51% in October, while saturated fatty acids (stearic acid, 18:0; palmitic acid, 16:0) decreased; (5) a temporal lag existed between peak fatty acid quality in POM and zooplankton: POM PUFA peaked at 60.22% in September and fell to 34.68% in October, whereas zooplankton EPA and ARA peaked in October, indicating delayed trophic transfer. These findings clarify seasonal dynamics and drivers of zooplankton fatty acids in a eutrophic plateau lake, providing a scientific basis for understanding nutrient transfer mechanisms in lake food webs.