Data describe the detection of female mitotypes from spectaclecase (Cumberlandia monodonta) and mucket (Ortmanniana ligamentina) freshwater mussel environmental DNA (eDNA) from water samples collected from the Big Piney River in Pulaski County, MO, U.S.A. Water samples were collected from 3 sites known to contain the target species. Sampling transects spanned from 100 meters upstream of the beds down to 3000 meters downstream at intervals of 500 meters, or as close as could possibly reach. Some transects were not reachable, therefore, no samples were collected from these transects. Sample collection occurred on a seasonal basis from June 2020 through April 2023, with the exceptions of autumn 2021 and winter 2022. Beyond these exceptions, sample collections were performed every 2-13 weeks (every 8 weeks on average). Samples were analyzed for the presence of eDNA by qPCR. Three different collection methods were performed; (1) 50 milliliter samples which were centrifuged, decanted, and concentrated pellet suspended for analysis, (2) glass fiber membranes were used to filter sample material, and (3), 1.2 micrometer polyether sulfone membranes were used to filter sample material. Parameters described in this data release include sample collection data, sample processing and extraction data, qPCR results, and associated quality assurance measurements from water samples.