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Molecular biomarkers preserved in lake sediments are increasingly used to develop records of past organism occurrence. When linked with traditional paleoecological methods, analysis of molecular biomarkers can yield new insights into the roles of herbivores and other animals in long-term ecosystem dynamics. We sought to determine whether fecal steroids in lake sediments could be used to reconstruct past ungulate use and dominant taxa in a small catchment in northern Yellowstone National Park. To do so, we characterized the fecal steroid profiles of a selection of North American ungulates historically present in the Yellowstone region (bison, elk, moose, mule deer, and pronghorn) and compared them with those of sediments from a small lake in the Yellowstone Northern Range. Analysis of a set of fecal steroids from herbivore dung (Δ5-sterols, 5α-stanols, 5β-stanols, epi5β-stanols, stanones, and bile acids) differentiated moose, pronghorn, and mule deer, whereas bison and elk were partially differentiated. Our results show that bison and/or elk were the primary ungulates in the watershed over the past c. 2300 years. Fecal steroid influxes reached historically unprecedented levels during the early and middle 20th century, possibly indicating high local use by ungulates. Comparison of fecal steroid influxes with pollen and diatom data suggests that elevated ungulate presence may have contributed to decreased forage taxa (Poaceae, Artemisia, and Salix), relative to long-term averages, and possibly increased lake production. Our results reflect past change within a single watershed, and extending this approach to a network of sites could provide much-needed information on past herbivore communities, use, and environmental influences in Yellowstone National Park and elsewhere., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wendt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)