In the Northwest Atlantic, Pandalus borealis (northern shrimp) serve as key mid-trophic consumers and prey for higher-trophic predators, including commercially important fish species. However, the impact of changing environmental conditions on trophic interactions and lipid storage in sub-Arctic ecosystems is not well understood. We employed biochemical tracers (fatty acids and stable isotopes) to investigate the trophic ecology and stage-specific nutritional condition of P. borealis across spatial and seasonal scales. A total of 68 different fatty acids (FAs) were identified in P. borealis tissues (i.e., muscle and eggs). The relative abundances of these FAs varied among sex, tissues, seasons, and fishing areas. Results revealed that P. borealis primarily fed on diatoms and zooplankton, with opportunistic feeding on sinking phytodetritus. Lipid composition showed strong seasonality, with storage triacylglycerols being the predominant lipid class. Ovigerous females exhibited the highest lipid concentrations and essential fatty acids, emphasizing the ecological importance of eggs as high-quality lipid sources. Additionally, total lipid content in eggs increased from spring to summer, highlighting vulnerability to shifts in seasonal primary production. This study underscores the significant seasonal variability in the nutritional status of P. borealis and the need to understand lipid dynamics to assess population resilience to environmental changes.