The Sturgeon chub (Macrhybopsis gelida) is a cyprinid fish native to the Missouri and Mississippi River basins of the U.S. Suspected long-term declines in the size of its distribution have prompted a review of its conservation status by the U.S. Fish and Wildlife Service, a process which depends on reliable methods to delineate the distribution and status of extant populations. To facilitate monitoring of Sturgeon chub populations, we developed a quantitative PCR assay to detect Sturgeon chub DNA in environmental samples. The assay consistently detected Sturgeon chub DNA in concentrations as low as 2 copies per reaction, and did not amplify DNA from non-target fish species that are sympatric in the upper Missouri River basin. Field tests of this assay with environmental samples successfully detected Sturgeon chub from sites known to be occupied. This assay offers an extremely sensitive methodology that can be applied to determine the range of Sturgeon chub, regardless of variation in habitat characteristics.