The depth selection behaviors of juvenile mysids, Mysis diluviana, exposed to different temperature and light gradients in the laboratory were compared to those observed in previous studies on adult mysids to determine whether mysid light and temperature preferences have an ontogenetic component. Juvenile mysids most strongly preferred 11 °C, which is 5 °C higher than those most preferred by adults, but both size classes completely avoided temperatures of 16 °C and greater. Juveniles and adults preferred light levels between 10-5 and 10-6 lx, or ∼10-7 and 10-8 “mylux” — a unit of brightness specific for mysid vision; however, juveniles did not display avoidance behaviors until 10-0.6 mylux (∼44 lx), compared to adults which avoid light levels of 10-3 mylux, or ∼0.1 lx, and above. Because the differences in preferences between juveniles and adults were more pronounced in relation to temperature than to light, we expect stronger vertical separation of mysid size classes during periods of the year when the water column is thermally stratified versus isothermal. We present limited field data and review past literature to support this hypothesis, as juvenile mysids are typically found higher in the water column than adults, especially during periods of the year when the water column is thermally stratified. This study is one of the first to demonstrate that ontogenetic differences in light and temperature preferences lead to habitat partitioning in a mysid species and highlights the often underreported importance of juvenile mysids to food web structure and function in open waters above the thermocline.