Proposed invasive carp barriers may threaten populations of migratory fishes in the St. Croix National Scenic Riverway and the Mississippi National River and Recreation Area by preventing movements between rivers needed to fulfill life history requirements. Moreover, reproducing populations of invasive carp could alter aquatic food webs and negatively affect mussels and migratory fishes. In this study, nonlethal chemical techniques were used to determine the trophic positions and migratory histories of lake sturgeon (Acipenser fulvescens) captured in the St. Croix and Mississippi rivers. Stable isotope analyses demonstrated differences in trophic position among sturgeon captured in different locations among the two rivers. Ratios of strontium to calcium (Sr:Ca) and barium to calcium (Ba:Ca) in collected water samples were characterized at six sampling locations among the two rivers. Laser ablation data obtained from cross sections of lake sturgeon pectoral fin rays indicated that lake sturgeon Sr:Ca values were different between rivers, but Sr:Ca values were similar for natal and capture locations of lake sturgeon captured in the same river. However, Sr:Ca data showed that some fish likely moved between the two rivers. Lake sturgeon captured in the Mississippi River had similar length-weight relations but higher length-at-age compared to lake sturgeon captured in the St. Croix River. Growth and Sr:Ca analyses provided evidence that the St. Croix and Mississippi rivers have separate populations of lake sturgeon that interact through migrations of individual fish between rivers. Results of this study will provide managers information about the feeding ecology and migration histories of lake sturgeon within these two park units and provide pre-invasion data for lake sturgeon should populations of invasive carp become established. In addition, laser ablation techniques presented here could be used to study other migratory fishes that serve as dispersal agents for at-risk mussel species.