Hemlock (Tsuga canadensis) forest of the eastern U.S. are in decline due to invasion by the exotic insect hemlock woolly adelgid (Adelges tsugae). Aquatic biodiversity in hemlock ecosystems has not been documented; thus the true impact of the infestation cannot be assessed. We compared ichthyofaunal assemblages and trophic structure of streams draining hemlock and hardwood forests by sampling first- and second-order streams draining 14 paired hemlock and hardwood stands during base flows in July 1997 at the Delaware Water Gap National Recreation Area of Pennsylvania and New Jersey. Over 1400 fish of 15 species and 7 families were collected, but hemlock and hardwood streams individually harbored only one to four species. Brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) were two to three times as prevalent in hemlock than hardwood streams. Insectivorous fishes occurred in significantly higher proportion in streams of hardwood (0.90) than hemlock (0.46) stands, while piscivores occurred more often in hemlock (0.85) than hardwood (0.54) stands. Functional (trophic) diversity of fishes in hemlock and second-order streams was numerically greater than that of hardwood and first-order streams. Species composition also differed by stream order and terrain type. Biodiversity is threatened at several levels within hemlock ecosystems at risk to the hemlock woolly adelgid in eastern U.S. forests.