The northern leatherside chub (Lepidomeda copei) is a cyprinid fish native to the Snake River, Green River, and Bonneville basins of the western United States. Population declines prompted the development of a multistate conservation agreement and strategy, which emphasized the need to reliably delineate its current distribution and monitor its status. To facilitate species monitoring, we developed a quantitative PCR assay to detect northern leatherside chub DNA in environmental samples. The assay consistently detected northern leatherside chub DNA in concentrations as low as 2 copies per reaction and did not amplify DNA of potentially sympatric fish species. The assay amplified a synthetic DNA template representing 3 congeneric species: White River spinedace (L. albivallis), Virgin spinedace, (L. mollispinis mollispinis), and Big Spring spinedace, (L. m. pratensis); however, none of these are sympatric with northern leatherside chub. Field tests of the assay accurately reproduced expected patterns of species occupancy.