Water quality programs in Connecticut and nationally have focused on restoring impaired waters, while modest attention has been allocated to healthy watersheds in the least disturbed condition. The objective of our study was to document the geographic location of least disturbed streams in Connecticut, describe the aquatic biota from these systems, and describe important environmental variables that may help explain the distribution of these biota. We used geographic information systems to select drainage basins by their natural attributes and by eliminating anthropogenic stressor variables in order to best approximate a least disturbed watershed condition in Connecticut. We then sampled the fish and macroinvertebrate communities, water chemistry, and associated GIS-derived watershed attributes to determine the variables that best described the sampled biota. We identified 30 least disturbed streams that had drainage areas <29 km², whose stream order ranged from 1–4, and that contained <4% total impervious cover in the upstream watershed. Least disturbed streams were generally located in three geographic areas of the state—northwest Connecticut, northeast Connecticut, and the central Connecticut valley—and were absent from the southern coast of Connecticut and southwestern Connecticut. Cluster analysis and nonmetric multidimensional scaling of macroinvertebrate taxa in the Orders Ephemeroptera, Plecoptera, and Trichoptera showed 3 macroinvertebrate stream classes, with 12 significant indicator species (P < 0.05). Drainage area, water temperature, alkalinity, hardness, chloride, ammonia, total nitrogen (TN), and total phosphorus (TP) may explain some of the differences in taxa between macroinvertebrate stream classes. Cluster analysis and nonmetric multidimensional scaling of fish species also showed three fish stream classes, with 9 significant indicator species (P < 0.05). Drainage area, stratified drift, dam density, water temperature, total suspended solids, alkalinity, hardness, ammonia, TN, and TP may explain some of the differences in species between fish stream classes. Ninety percent of the least disturbed streams sampled contained Salvelinus fontinalis (Brook Trout), which can be considered a sentinel fish species for small, least disturbed streams in Connecticut.