We examined vertical, longitudinal and seasonal variation in the abundance, taxa richness and community composition of the epibenthic and hyporheic macrobenthos at Elklick Run, a first- through fourth-order stream continuum in the central Appalachian Mountains in West Virginia. We sampled the macrobenthos at three levels (0–10 cm, 10–20 cm, 20–30 cm) below the stream bed using baskets filled with natural substratum. We quantified environmental factors including interstitial organic matter, fine sediment, water velocity and dissolved oxygen. Abundance, taxa richness and the relative abundance of streamlined taxa decreased with depth into the substratum. Interactions between depth into the substratum and site and between depth and season, were caused by a winter spate that reduced the abundance and taxa richness of the benthos in the upper layer of the hyporheic habitat (0–10 cm), but only at downstream sites. There was broad overlap in community structure among depths into the hyporheos, and virtually no taxa increased in absolute abundance with depth into the substratum or were restricted to hyporheic habitat. Abundance and taxa richness varied more with depth into the substratum than among sites or among seasons. However, both epibenthic and hyporheic community structure varied much more among seasons than among sites or among depths. The hyporheic fauna resembled the epibenthic fauna less at downstream sites where there was a more specialized epibenthic fauna including more streamlined taxa, filterers and scrapers. Abundance and taxa richness of the macrobenthos was positively correlated with interstitial flow, especially at upstream sites in Fall when stream flow was lowest, water temperature was high and interstitial dissolved oxygen concentration was low. The coefficient of variation in hyporheic abundance, taxa richness and community composition decreased with increasing stream order, suggesting decreased spatial variability in interstitial habitat suitability at downstream sites.