Understanding how biological communities vary across regions with distinct environmental contexts is central to stream bioassessment, yet most frameworks emphasize taxonomic structure while giving more limited attention to functional organization. Using a coordinated regional dataset from 90 wadeable streams across three regions of the Chesapeake Bay watershed (Valley & Ridge, Piedmont, and Coastal Plain), we evaluated how macroinvertebrate structural and functional indicators vary among regions and biological condition classes defined by the Chesapeake Basin-wide Benthic Index of Biotic Integrity (B-IBI). Structural organization was characterized using taxonomic composition and diversity metrics commonly incorporated into multimetric indices (taxonomic richness and Shannon diversity). Functional composition was quantified using density- and biomass-weighted community-weighted means (CWMs) of traits associated with environmental sensitivity (response traits), ecosystem functions and energy pathways (effect traits), and organism–habitat and resource interactions (linking traits), organized within a response–linking–effect (REL) framework. Functional organization beyond composition was evaluated using functional evenness (FEve), Rao’s quadratic entropy (RaoQ) and functional redundancy (FR), which describe the distribution, dispersion and overlap of traits within communities. Because the B-IBI is grounded in taxonomic composition and tolerance, we evaluated whether structural indicators and response traits aligned with biological condition classes, and whether linking traits, effect traits, and functional organization exhibited stronger region- and weighting-dependent patterns. Relationships between macroinvertebrate indicators and B-IBI varied by region, weighting scheme and REL domain and were not consistently monotonic. Structural indicators showed limited or inconsistent correspondence with B-IBI across regions. However, FEve, RaoQ, FR and selected REL composites and trait CWMs related to habitat association, emergence timing and body size showed more consistent region-specific associations with B-IBI. Consequently, streams with similar B-IBI condition classes often differed in functional organization and energetic structure, while streams with different B-IBI scores also exhibited comparable functional responses. Together, these patterns indicate that biological condition classes capture important variation in community structure but do not correspond to a single, uniform trajectory of functional organization. Similar B-IBI scores can arise from distinct functional configurations that differ in trait composition, redundancy, and energetic structure, reflecting differences in how stream communities may buffer disturbance and reorganize following environmental change.