1. It is essential to establish a baseline in studies using stable isotopes to interpret trophic relationships across ecosystems and through time. Studies in freshwater ecosystems struggle to quantify baseline stable isotopes due to difficulties collecting representative samples, particularly from pelagic habitats. We assessed temporal and spatial variation in δ 13 C, δ 15 N, and δ 34 S in a commonly used pelagic baseline, seston (n = 156), in Lake Erie to understand mechanisms that correlate with baseline stable isotope dynamics in large lakes. Seston contains a wide range of material which can confound stable isotope interpretation, and we examined the utility of element content and ratios to account for variation in sample source.2. Seston was collected in each of the three basins of Lake Erie from May to October in 2017-2019 at nearshore (<10 m depth) and offshore (>10 m depth) sites.General linear models were conducted on each stable isotope (δ 15 N, δ 13 C, and δ 34 S) and sample composition (variables: %N, %C, %S, C:N, C:S, and N:S) to assess how basin, month, and collection year influenced seston stable isotopes and composition.