Aquatic and terrestrial ecosystems vary in the abundance and composition of polyunsaturated fatty acids (PUFAs). Omega 3 (ω3) PUFAs such as EPA (Eicosapentaenoic acid) and DHA (Docosahexaenoic acid) are abundant in aquatic ecosystems but extremely scarce in terrestrial ecosystems. As a result, riparian predators, such as spiders, depend on aquatic subsidies such as emergent insects to meet their requirement for these ω3 PUFAs. In alpine ecosystems, however, these resources can be seasonally variable, with insect emergence occurring predominantly in the warmer months and largely ceasing in winter. This study aims to understand the spatial and temporal variation of ω3 PUFAs in riparian spiders, with particular focus on EPA and DHA. We quantified total PUFA, EPA, DHA as well as the ratio of omega 3 to omega 6 (ω6) fatty acids in riparian spiders collected at varying distances (0-80 meters) from alpine creeks in spring, summer and autumn to understand the cross-ecosystem transfer of PUFA. Total ω3 PUFA and ω3:ω6 was estimated using three ω3 PUFAs, EPA, DHA and α-linolenic acid (ALA) and three ω6 PUFAs, γ-Linolenic acid (GLA), Linoleic acid (LIN), and Arachidonic acid (ARA). Results revealed that EPA and DHA represented on average 30% of total PUFAs and 80% of ω3 PUFAs in riparian spiders. Total ω3 PUFA, EPA, and DHA concentrations, and ω3:ω6 ratio declined with increasing distance from the creek, closely mirroring the decline in emergent insect abundance. In contrast, non-aquatically derived fatty acids (ALA and ω6 PUFA) showed no significant spatial patterns. Seasonal variation was significant for DHA but not EPA concentration, indicating seasonal changes in their availability to spiders. These results highlight the important contribution of aquatic-derived PUFAs to the fatty acid profiles of riparian spiders.