Determining the natal origins of fish can provide critical information for fisheries management and conservation efforts. Differences in forage among fish rearing locations may result in unique isotopic signatures in the fish tissue, and historically archived isotope signatures in fish eye lenses may be useful in differentiating rearing sources. The objective of this study was to investigate the feasibility of using isotopic composition of fish eye lenses to discern the known origin of fall, advanced fingerling age-0 Walleye (Sander vitreus) reared in a recirculating aquaculture system (RAS), hatchery ponds, and a natural lake. We collected 10 RAS-reared, pellet-fed Walleye from Cleghorn fish hatchery in Rapid City, South Dakota, 10 pond-reared Walleye from Blue Dog hatchery in Waubay, South Dakota, and 10 wild-caught Walleye from Clear Lake near Sioux Falls, South Dakota. Layers were delaminated to the central core from one eye lens to assess change in carbon (δ13C) and nitrogen (δ15N) isotope signatures over time, and whole eye lenses from the second eye were pulverized and analyzed for δ13C, δ15N, and sulfur (δ34S). The RAS-reared Walleye had highly precise isotope signatures among individuals and enriched δ34S signatures, whereas pond-read Walleye had depleted δ13C and δ15N eye lens core signatures. Isotopic signatures of δ13C and δ15N stayed consistent over time (across lens layers) for RAS-reared Walleye; slight increases in δ15N occurred for both pond-reared and wild-caught Walleye, but a substantial increase in δ13C occurred in pond-reared Walleye lens layers over time. However, we observed no overlap in Bayesian ellipses among the rearing sources when comparing δ13C and δ15N core, layer, or whole eye lens signatures and for δ13C and δ34S whole lens signatures. The results of this study demonstrate that eye lens stable isotope analysis may be a useful management tool for identifying the stock contribution of RAS and pond-reared fishes.