Data associated with the manuscript "Stable isotopes and diet metabarcoding reveal trophic overlap between native and invasive Banded Killifish (Fundulus diaphanus) subspecies." by Jordan H. Hartman, Mark A. Davis, Nicholas J. Iacaruso, Jeremy S. Tiemann, Eric R. Larson. For this project, we sampled six locations in Michigan and Illinois for Eastern and Western Banded Killifish and primary consumers. Using stable isotope analysis we found that Eastern Banded Killifish had higher variance in littoral dependence and trophic position than Western Banded Killifish, but both stable isotope and gut content metabarcoding analyses revealed an overlap in the diet composition and trophic position between the subspecies. This dataset provides the sampling locations, accession numbers for gut content metabarcoding data from the National Center for Biotechnology Information Sequence Read Archive, the assignment of each family used in the gut content metabarcoding analysis as littoral, pelagic, terrestrial, or parasite. and the raw stable isotope data from University of California Davis.