Better knowledge of upstream migratory patterns of spawning Sea Lampreys Petromyzon marinus, an invasive species in the Great Lakes, is needed to improve trapping for population control and assessment. Although trapping of adult Sea Lampreys provides the basis for estimates of lake-wide abundance that are used to evaluate the Sea Lamprey control program, traps have only been operated at dams due to insufficient knowledge of Sea Lamprey behavior in unobstructed channels. Acoustic telemetry and radiotelemetry were used to obtain movement tracks for 23 Sea Lampreys in 2008 and 18 Sea Lampreys in 2009 at two locations in the Mississagi River, Ontario. Cabled hydrophone arrays provided two-dimensional geographic positions from acoustic transmitters at 3-s intervals; depth-encoded radio tag detections provided depths. Upstream movements occurred at dusk or during the night (2015–0318 hours). Sea Lampreys were closely associated with the river bottom and showed some preference to move near banks in shallow glide habitats, suggesting that bottom-oriented gears could selectively target adult Sea Lampreys in some habitats. However, Sea Lampreys were broadly distributed across the river channel, suggesting that the capture efficiency of nets and traps in open channels would depend heavily on the proportion of the channel width covered. Lack of vertical movements into the water column may have reflected lamprey preference for low water velocities, suggesting that energy conservation was more beneficial for lampreys than was vertical searching in rivers. Improved understanding of Sea Lamprey movement will assist in the development of improved capture strategies for their assessment and control in the Great Lakes.