environmental dna (edna) detection has been proposed as a potential method for determining the presence, abundance, and biomass of species within aquatic habitats. however, it is unclear what spatial scale(s) edna detections represent in lotic systems due to uncertainties surrounding its production and persistence. this study had two objectives: (1) to determine at what spatial scale(s) (length of stream segments) could edna concentration be related to the abundance and/or biomass of brook trout (salvelinus fontinalis), and (2) to determine if the relationship between edna concentration and brook trout abundance/biomass could be improved by accounting for the contribution of edna originating upstream of investigated stream segments. concentrations of edna were related to the abundance of brook trout at the 25m (r2 = 0.22), 50m (r2 = 0.18), and 100m (r2 = 0.42) spatial scales, and to the biomass of brook trout at the 25m spatial scale (r2 = 0.06), suggesting that edna concentrations can represent brook trout abundance across numerous spatial scales. variability observed in these relationships across spatial scales was likely due to (1) the presumably heterogeneous distribution of brook trout within stream segments causing different concentrations of edna to be detected for similar abundances and biomasses, and (2) the contribution of edna from upstream of examined stream segments to the detected edna concentrations. concentrations of edna were calibrated to 25m stream segments by accounting for the contribution of upstream edna assuming either the constant or conditional deposition of edna. both calibration methods improved the relation of edna concentrations to the abundance and biomass of brook trout within the 25m segments (abundance, r2 = 0.327 and r2 = 0.336 for constant and conditional calibration respectively). however, both methods incorrectly estimated the persistence and contribution of upstream edna on occasion, suggesting that additional investigation is required before these methods can be reliably and confidently implemented to assess the abundance and biomass of brook trout in lotic environments.