Environmental DNA (eDNA) offers a novel approach to supplement traditional surveys and provide increased spatial and temporal information on species detection, and it can be especially beneficial for detecting at risk or threatened species with minimal impact on the target species. The transport of eDNA in lotic environments is an important component in providing more informed descriptions of where and when a species is present, but eDNA transport phenomena are not well understood. In this study, we used species-specific assays to detect eDNA from two federally endangered mussels in two geographically distinct rivers. Using the eDNA concentrations measured from field samples, we developed a one-dimensional (1D) hydrodynamic transport model to predict the downstream fate and transport of eDNA. We detected eDNA from both federally endangered mussels across several seasons and flow rates and up to 3.5 km downstream from the source populations, but the detection rates and eDNA concentrations were highly variable across and within rivers and study reaches. Our 1D transport models successfully integrated the variability of the eDNA field samples into the model predictions and overall model results were generally within ±1 standard error of the eDNA field concentration values. Overall, the results of this study demonstrate the importance of optimizing the spatial locations from where eDNA is collected downstream from a source population, and it highlights the need to improve understanding on the shedding mechanisms and magnitude of eDNA from source populations and biogeomorphic processes that influence eDNA transport.