The relationships between stream flow regime and macroinvertebrate diversity, community structure, and functional feeding groups (FFG) were examined to determine if the biodiversity and macroinvertebrate fauna of non-perennial streams are significantly different from those of perennial streams. The study was conducted in northeastern Massachusetts at headwater stream sites of varying flow permanence (perennial, intermittent, and ephemeral). ANOVA confirmed no significant difference in Shannon-Wiener diversity (H′) between stream types, demonstrating that non-perennial streams maintain diverse and even macroinvertebrate communities. Whereas taxa richness was equal among intermittent and perennial sites, ephemeral richness was lower due to their significantly lower riffle richness. Qualitatively, two non-perennial sites were higher in grand total H′ diversity and taxa richness than perennial sites. Community structure was also related to flow regime, as hierarchical cluster analysis (HCA) based on taxa presence produced three distinct groups consistent with stream type, and FFG analysis provided further evidence of distinct communities, with a transition in FFGs from perennial to ephemeral sites. This study concludes that non-perennial streams are biologically diverse and maintain distinct benthic communities and therefore contribute to stream biodiversity and river ecosystems.