Spectaclecase (Margaritifera monodonta) is a federally endangered freshwater mussel species that has experienced a 55% reduction in range (USFWS 2014) and is currently concentrated in three rivers in the Midwest of the United States (Gasconade, Meramec Rivers, MO, and St. Croix River, WI). Its preference for living under large rocks and boulders has limited detection of new populations by traditional survey methods. Environmental DNA technology has been used to detect invasive and rare species, but its use for detection of rare, benthic-dwelling species in large flowing systems has been limited. Here, we propose using environmental DNA to identify presumable sites for discovery of M. monodonta. We designed a M. monodonta-specific qPCR assay and tested it using M. monodonta-housed tank water and water samples from two known mussel beds on the St. Croix River and three known mussel beds on the Mississippi River. We observed higher overall detection rate on the St. Croix River (30.2%) compared to the Upper Mississippi River (0.60%). We also observed higher eDNA detection rates (73.3-93.1%) in 2018 for samples collected during the larval release period in May compared to samples collected in August after reproduction stopped (55.6-70.8%) on the St. Croix River. We tested samples collected at three distances downstream of the two mussel beds found in the St. Croix River, but we did not observe a significant effect of distance on our detection rates. However, we did observe greater detection rates for samples collected near the bottom compared to at the surface. Our results indicate that this novel qPCR assay can successfully detect M. monodonta eDNA and could be utilized to rapidly screen locations to guide intensive physical searches for populations in riverine systems.