Introduced lake trout (Salvelinus namaycush) threaten the native cutthroat trout (Oncorhynchus clarki bouvieri) population in Yellowstone Lake, Yellowstone National Park, Wyoming. A reduction in availability of cutthroat trout may affect fish predators negatively, especially during spawning season. In this study we examined the importance of spawning cutthroat trout to a fish specialist—the North American river otter (Lontra canadensis). We measured scent-marking rates at 87 otter latrine sites on Yellowstone Lake and its tributary streams throughout the cutthroat trout spawning period and identified prey in scats collected at these sites. Based on fecal deposition rates, otters were more active on spawning streams and less active on Yellowstone Lake during the height of cutthroat trout spawning, with a return to elevated activity on the lake after spawning had ended. Cutthroat trout appeared to be the most common prey item in otter scats throughout the study, based on 515 samples identified to the family level and 110 samples analyzed to the species level. Overall, trout occurred in 72% and longnose suckers (Catostomus catostomus) in 43% of otter feces. Suckers were more prevalent than trout in otter scats only on tributary streams, toward the end of cutthroat trout spawning. Introduced lake trout, which inhabit deep water and are largely inaccessible to otters, occurred in less than 5% of otter scats. River otters in the Yellowstone Lake ecosystem appear to be relatively restricted in their diet and are heavily dependent on cutthroat trout. Our findings suggest that continued declines in the cutthroat trout population could negatively impact otters, potentially disrupting their role in linking aquatic and terrestrial ecosystems in and around Yellowstone Lake.