Margaritifera monodonta, or the Spectaclecase Mussel, is a federally endangered freshwater mussel species that has experienced a 55% reduction in range and is currently concentrated in 3 rivers in the Midwest region of the United States (Gasconade and Meramec rivers, Missouri, and St Croix River, Wisconsin). The detection of new populations by traditional survey methods has been limited because these mussels tend to occur under large rocks and boulders. Environmental DNA (eDNA) 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 eDNA to assess known populations of M. monodonta. We designed a M. monodonta-specific quantitative polymerase chain reaction (qPCR) assay and tested it using water samples from multiple M. monodonta housing tanks, water samples from 2 known mussel beds on the St Croix River, and water samples from 3 known mussel beds on the Mississippi River. We observed higher overall eDNA detection rates 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 the reproductive period had ended (55.6–70.8%) on the St Croix River. We tested samples collected at 3 distances downstream from the 2 mussel beds found in the St Croix River, but we did not observe a substantial 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 used to rapidly screen locations to guide intensive physical searches for populations in riverine systems.