<p>Detecting and monitoring imperiled freshwater mussels is challenging due to their cryptic life history, low abundances, and habitat fragmentation. Environmental DNA (eDNA) offers a sensitive and non-invasive approach to detect freshwater mussels sight-unseen and can be a powerful tool for conservation and management of at-risk species. Here we describe the development and validation of quantitative PCR (qPCR) assays for the detection of three unionid species of conservation concern and evaluate sampling methodologies that may influence the detection of these species via eDNA. TaqMan assays were developed for the Federally endangered northern riffleshell (<i>Epioblasma rangiana</i>), snuffbox (<i>Epioblasma triquetra</i>), and the critically imperiled brook floater (<i>Alasmidonta varicosa</i>). Primer and probe sets were tested for specificity against DNA from 74 Unionidae, including co-occurring species. In vitro tests consistently detected focal species at environmentally relevant concentrations. No cross-amplifications were observed for any of the 74 non-target species tested by the respective eDNA assays. The utility of each qPCR assay was evaluated against eDNA samples collected from streams across the Mid-Atlantic United States, where target species’ presence or absence is documented. The assays successfully detected each focal species when present, and no eDNA detections were observed when species were absent. Based on occupancy modeling, detection rates for each of the assays are greater than 95% when the appropriate level of effort is applied. The eDNA assays presented herein provide an efficient and non-invasive means to inventory and monitor rare freshwater mussel species and can be used to guide more localized, traditional monitoring efforts.</p>