Fatty acid and stable isotope signatures allow researchers, managers, and policy makers to better understand food webs, food sources, and trophic relationships.Research in marine and lentic systems has indicated that the variance of these biomarkers can exhibit substantial differences across spatial and temporal scales, but this type of analysis has not been completed for large river systems. Our objectives were (a) to evaluate variance structures for fatty acids and stable isotopes (i.e., δ 13 C and δ 15 N) of seston, threeridge mussels, hydropsychid caddisflies, gizzard shad, and bluegills across spatial scales (10s-100s of km) in large rivers of the Upper Mississippi River Basin, USA, that were sampled annually for 2 years and (b) to evaluate the implications of this variance on the design and interpretation of trophic studies. The greatest variance for both stable isotopes was present at the largest spatial scale for all taxa (except seston δ 15 N), indicating that these isotopic signatures are responding to factors at a larger geographic level rather than being influenced by local-scale alterations. Conversely, the greatest variance for fatty acids was present at the smallest spatial scale (i.e., among individuals) for all taxa except caddisflies, indicating that the physiological and metabolic processes that influence fatty acid profiles can differ substantially between individuals at a given site. Our results highlight the need to consider the spatial partitioning of variance during sample design and analysis, as some taxa may not be suitable for use in assessing ecological questions at larger spatial scales. KEYWORDS carbon stable isotope, fatty acid, food webs, lotic, multivariate variance, nitrogen stable isotope