Springs are commonly used as low-cost monitoring locations to assess groundwater quality and long-term trends. However, spring waters in many settings are a mixture of groundwater sources that range in physical properties and water chemistry. The objective of this work was to determine water sources of springs emerging from the North American midcontinent Cambrian-Ordovician aquifer system at a fish hatchery near Lanesboro, Minnesota (USA), and compare and contrast the sources to shallower and deeper sources. The hydrology of the Lanesboro State Fish Hatchery has been studied for decades using a combination of dye tracing, thermal monitoring, geochemical sampling, and nearby borehole and outcrop observations. Previous studies are integrated with recently collected dye tracing results and geochemical data to develop a comprehensive conceptual model of groundwater flow. Dye trace findings and geochemistry indicate well-developed karst and bedrock fractures in shallowly buried unconfined carbonate formations are important transport pathways to convey anthropogenically influenced waters from the land surface to the hatchery springs. However, borehole dye traces, thermal monitoring, continuous nitrate monitoring, and mixing calculations show that a deeper confined siliciclastic aquifer is responsible for delivering relatively pristine water that accounts for about half of hatchery spring flux. Characterization of the hatchery’s groundwater systems provides fishery managers with information to protect this vital resource and improved context to interpret water-quality-monitoring data that track agricultural contaminants. The methods and results of this study may be widely applicable across a large extent of the Cambrian-Ordovician aquifer system, and to multiaquifer sedimentary bedrock systems elsewhere.