The use of stable isotopes has recently grown in studies of trophic structure and the recruitment and migration patterns of consumers. This type of analysis allows the flow of organic matter and trophic relationships to be outlined within complex systems. Although multiple stable isotope analysis is useful in distinguishing linkages between sources and consumers, its efficacy is contingent on the isotopic similarity within each source and the distinctiveness of producer isotope values. As part of a study investigating juvenile Chinook salmon (Oncorhynchus tshawytscha) food webs in the Columbia River estuary we examined the isotopic (δ¹³C, δ¹⁵N, and δ³⁴S) composition of five prominent primary producers. We sought to examine statistical variability of primary producer isotope values and its effects on differentiation between sources. We found that within-group isotopic variability occurs at different scales, related to the heterogeneous landscape in which producers grow. Aquatic and wetland vascular plants displayed the greatest range in isotopic composition while benthic algae and particulate organic matter were more constrained in their isotope signatures. When examining differences between groups we found that although δ¹³C was the most variable isotope ratio, it was also the most useful in distinguishing sources, especially benthic producers compared to water column and emergent producers. Signatures of δ¹⁵N and δ³⁴S were most useful in differentiating benthic algal and vascular marsh plant groups. Isotopic composition along with chlorophyll and elemental composition was also useful in distinguishing phytoplankton samples from particulate organic matter (POM) samples. The type and extent of isotopic variability revealed by this study will help inform future food web studies using isotopes to characterize trophic linkages in large estuaries such as the Columbia River estuary.