Diel vertical migration (DVM) is critical for moving energy and nutrients between surface and deep waters. Mysis sp. (Crustacea: Mysidae) facilitates this process by serving as predator and prey in both benthic and pelagic habitats. Mysis can also exhibit partial DVM (pDVM), where some individuals do not migrate into the pelagia at night or to the benthos during the day. However, whether Mysis pDVM is a fixed (i.e., same individuals migrate) or random (i.e., random individuals migrate) behavior remains unclear. To evaluate that, we tested whether Mysis exhibit pDVM and niche partitioning in Lake Superior by collecting Mysis from benthic and pelagic habitats day and night across depths ranging from 50 to 250 m and estimating their isotopic niche size (δ¹³C, δ¹⁵N; Corrected Standard Ellipse Area − SEA_c) and overlap among three life stages. At the population level, Mysis exhibited fixed pDVM structured by life stage. Benthic Mysis (12.9 ± 3.2 mm, mean ± SD) were larger than pelagic Mysis during night (9.6 ± 3.6 mm) and day (8.5 ± 3.6 mm). Adult Mysis (> 15 mm) had larger SEA_c (1.8 ± 0.4 ‰²) compared to juveniles (< 10 mm; 0.3 ± 0.1 ‰²) and sub-adults (10–15 mm; 0.7 ± 0.2 ‰²), and their isotopic niche did not overlap with smaller life stages. Adults exhibited random pDVM (i.e., high isotopic niche overlap), whereas juveniles exhibited fixed pDVM (i.e., low overlap). Our observations indicate complex behaviors across and within Mysis life stages, likely due to varying pressures including size-selective predation, light and temperature thresholds, and nutritional requirements. Consequently, the benthic behavior of adult Mysis needs to be considered in monitoring programs.