Effective methods to control invasive Sea Lampreys Petromyzon marinus in the Laurentian Great Lakes often rely on knowledge of the timing of the Sea Lamprey spawning migration, which has previously been characterized using data gathered from traps. Most assessment traps are located many kilometers upstream from the river mouth, so less is known about when Sea Lampreys enter spawning streams and which environmental cues trigger their transition from lakes to rivers. To decide how to develop barriers and traps that target Sea Lampreys when they enter a stream, the stream entry of Sea Lampreys into a Lake Huron tributary during 2 years was assessed using dual-frequency identification sonar (DIDSON). Sea Lampreys entered the stream in low densities when temperatures first reached 4°C, which was up to 6 weeks and a mean of 4 weeks earlier than when they were first captured in traps located upstream. The probability of stream entry was significantly affected by stream temperature and discharge, and stream entry timing peaked when stream temperatures rose to 12°C and discharge was high. Examination of the entry at a finer temporal resolution (i.e., minutes) indicated that Sea Lampreys did not exhibit social behavior (e.g., shoaling) during stream entry. Our findings indicate that Sea Lampreys may be vulnerable to alternative trap types near river mouths and hydraulic challenges associated with traditional traps. Also, seasonal migration barriers near stream mouths may need to be installed soon after ice-out to effectively block the entire adult Sea Lamprey cohort from upstream spawning habitat.