Understanding the habitat needs of fish and how these requirements may change seasonally over a 24-h period is important, especially for highly managed sport species. Consequently, we examined the diel and seasonal habitat use of four juvenile salmonid species in streams in the Lake Ontario watershed. For juvenile Atlantic salmon Salmo salarand juvenile rainbow trout Oncorhynchus mykiss, differences in day versus night habitat use were more profound than seasonal differences. Observed differences in day versus night habitat for all species and age classes were mainly due to the use of less object oriented cover at night and to a lesser extent to the use of slower velocities and smaller substrate at night. Seasonal differences in habitat use were also observed, likely due to increased fish size, and included movement to deeper and faster water and the use of larger substrate and more cover from summer to winter. Different habitat variables were important to individual species. Juvenile Atlantic salmon were associated with higher water velocities, juvenile rainbow trout with larger substrate and more cover, and subyearling Chinook salmon O. tshawytscha and subyearling coho salmon O. kisutch with small substrate and less cover. Our observations demonstrate that habitat partitioning occurs and likely reduces intraspecific and interspecific competition which may increase the potential production of all four species in sympatry. Consequently, these findings provide important information for resource managers charged with managing, protecting, and enhancing Great Lakes tributaries where all or some of these species occur.