Otolith microchemistry is a commonly used tool for stock discrimination in fisheries management. Two key questions remain with respect to its effectiveness in discriminating among river-spawning populations. First, do larvae remain in their natal river long enough for their otoliths to pick up that systemâ s characteristic chemical signature? Second, are larval otolith microchemical differences between natal rivers sufficiently large to overcome spatiotemporal variation in water chemistry? We quantified how larval age, the ratio of ambient strontium to calcium concentrations (Sr:Ca), and water temperature influence otolith Sr in both lab-reared and wild-collected Lake Erie walleye (Sander vitreus). Otolith microchemistry shows promise as a spawning stock discrimination tool, given that otolith Sr in larval walleye: 1) is more strongly influenced by ambient Sr:Ca than by temperature; 2) reflects Sr:Ca levels in the natal environment, even in larvae as young as 2 d; and 3) can effectively discriminate between larvae captured in two key Lake Erie spawning tributaries, even in the face of short larval river-residence times and within-year and across-year variation in ambient Sr:Ca. ; The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author.