Pteronarcys stoneflies, which are traditionally considered shredders in eastern North America, inhabit second- through seventh-order streams in the Little Tennessee River (LTR) drainage basin. Because very little coarse allochthonous particulate organic matter occurs in the relatively large (sixth- and seventh-order) LTR, we analyzed the gut contents of five individuals from each of five sites and two dates (August and December) (n = 50) along a fourth- through seventh-order stretch of this stream continuum. Pteronarcids consumed significantly different percentages of some food items between sampling dates. Pteronarcys progressively consumed significantly more diatoms (from <1% of gut contents at stream order 4 to 46% at stream order 7) and significantly less detrital material (from >90% of gut contents at stream order 4 to ca. 50% at stream order 7) from fourth- to seventh-order sites, and the amount of Pteronarcys production attributable to each of these food items shifted significantly along the stream continuum. This diet shift raised the obvious question: Does the shift reflect changing Pteronarcys species composition or is only one species present throughout the continuum? We conducted an allozyme electrophoretic analysis on 62 pteronarcids from the LTR drainage basin using five diagnostic gene loci for eastern USA Pteronarcys species. We determined that three apparent species occur along this continuum. Pteronarcys species A inhabits the upstream sites (fourth- through sixth-order), Pteronarcys species C the downstream sites (sixth- and seventh-order), and Pteronarcys species B the mid-reach site where A and C co-occur (sixth-order). Based on their diet and distribution, Pteronarcys sp. A and P. sp. C exhibit different patterns of food consumption along the continuum, with the former consuming mainly detritus and the latter consuming detritus and diatoms.