The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish once abundant throughout coastal basins of western North America that has suffered dramatic declines in the last century due primarily to human activities. Here, we describe the development of an environmental DNA (eDNA) assay to detect Pacific lamprey in the Columbia River basin. The eDNA assay successfully amplified tissue derived DNA of Pacific lamprey collected from 12 locations throughout the Columbia River basin. The assay amplifies DNA from other Entosphenus species found outside of the Columbia River basin, but is species-specific within this basin. As a result, the assay presented here may be useful for detecting Entosphenus spp. in geographic range beyond the Columbia River Basin. The assay did not amplify tissue or synthetically derived DNA of 14 commonly sympatric non-target species, including lampreys of the genus Lampetra, which are morphologically similar to Pacific lamprey in the freshwater larval stage.