Environmental DNA (eDNA) is an effective tool for the detection of elusive or low-density aquatic organisms. However, it has infrequently been applied to mammalian species. North American river otters (Lontra canadensis) are both broad ranging and semi-aquatic, making them an ideal candidate for examining the uses of eDNA for detection of mammals. We developed a speciesspecific assay for detection of North American river otters using eDNA. The assay was tested for specificity against closely-related mustelids native to western North America, and was validated through testing environmental samples.