Lack of information about the rates and sources of population‐specific mortality and habitat use during nonspawning periods has impeded the restoration of lake sturgeon Acipenser fulvescens. Using eight microsatellite loci and mixed‐stock analyses, we estimated the proportional contributions of spawning populations from throughout the Lake Michigan basin (n = 5) to the fall sport fishery in the lower Menominee River, Wisconsin. We compared estimates of harvest composition with estimates from collections made in adjacent open‐water habitats in Green Bay. The analyses revealed that 81% (90% confidence interval [CI] = 72.9–89.3%; N = 104) of harvested individuals originated from the Menominee River; all of the bycaught fish originated in adjacent streams. The harvest composition estimates differed significantly from those of open waters immediately offshore (26.7% Menominee River; 90% CI = 9.7–44.8%; N = 36) and across Green Bay (27% Menominee River; 90% CI = 19.5–34.7%; N = 214), indicating that the harvest was not a random sample from across the basin. The harvest composition estimates were not consistent with the estimates of individuals in prespawning condition (females = 50%; males = 83%), suggesting that not all of the harvested fish were staging for spring spawning. The contributions of nontargeted and numerically depressed populations to the fishery are of management concern given efforts to rehabilitate populations. Spatially restricted harvests during nonbreeding periods may not protect numerically depressed populations originating in nearby streams.