Environmental DNA (eDNA) is potentially a non-invasive, cost-effective and efficient method for monitoring aquaculture and commercial fisheries populations to inform sustainable fisheries
management practices. eDNA tools must be thoroughly ground truthed to determine
best practices for their appropriate application. Using sea scallop aquaculture farms and a vertically-stratified sampling design above a wild sea scallop bed in Penobscot Bay, Maine, we evaluated the temporal and spatial variability in scallop eDNA signal. The available scallop qPCR probe and primers successfully detected scallop eDNA on scallop aquaculture farms, above a wild well-characterized, deeper scallop bed, and at a site lacking sea scallops and established high temporal and spatial variation in this signal. Seasonal gene copy number per liter seawater maxima on sea scallop farms did not occur after peak scallop spawning, as indicated by GSI values, and did not occur in tandem with maximum counts of bivalve larvae with one exception of one farm site. Sea scallop eDNA was detected at all depths above a wild scallop bed and at a site lacking scallops, indicating that transport of eDNA and quantifying stochasticity in ‘background’ signals is an important consideration in future studies. The scallop eDNA signal increased at both wild population sites and across depths after maximum GSI were observed during the time of assumed peak larval presence from 30-45 days after spawning. The high spatial and temporal variability in scallop DNA detection supports the need for carefully constructed sampling designs that are informed by organismal life history traits and patterns and the physical oceanographic characteristics of local waters to best apply eDNA tools to monitoring commercially important species.