In 2014 North American River Otters expanded up the North Fork of the Merced River into previously and historically unoccupied Yosemite Valley. Their range expansion posed the questions: 1) What are river otters preying upon in Yosemite Valley? 2) Could invasive Signal Crayfish provide a prey subsidy that promotes the upward elevational expansion of river otter populations into Yosemite Valley? To answer these questions, otter scats were collected and prey DNA present in the scats was amplified using a metabarcoding approach. The diet data revealed that invasive Signal Crayfish were present in 100% of scats collected and native Sacramento Sucker was present in only 16% of the scats. These results indicate that invasive Signal Crayfish are the most important diet species in the river otter diet, and the invasion of the Yosemite Valley by Signal Crayfish may have provided essential nutritional resources that facilitated upward elevational range expansion by river otters from Central Valley drainages. This study adds to the body of literature supporting the use of next-generation sequencing technologies and environmental DNA for addressing the ecology of species and ecosystem dynamics in the natural world.