American eel Anguilla rostrata populations are declining over much of their native range. Since American eels spend extended periods in freshwater, understanding their habitat requirements while freshwater residents is important for the management and conservation of this species. As there is little information on American eel habitat use in streams, the ontogenetic, diel, and seasonal habitat use as well as habitat selectivity of three size groups (i.e. ≤199 mm total length, 200–399 mm, ≥400 mm) of eel were examined in a tributary of the Hudson River. American eels in Hannacroix Creek exhibited ontogenetic, diel, and seasonal variation in habitat use as well as habitat selection. During both summer and autumn all sizes of American eels used larger substrate and more cover during the day. American eels ≤199 mm exhibited the strongest habitat selection, whereas eels 200–399 mm exhibited the least. During the autumn all sizes of American eels occupied slower depositional areas where deciduous leaf litter accumulated and provided cover. This may have important implications for in-stream and riparian habitat management of lotic systems used by American eel.