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Stream macroinvertebrates were collected from four intermittent tributaries and two perennial sites within the Kings Creek basin, Konza Prairie Biological Station (KPBS) near Manhattan, KS, USA. The objectives of this study were to assess the roles of disturbances (floods and drying) and refugia on benthic and colonization (drift and aerial) assemblage composition over two years among sites with contrasting hydrologic regimes. Benthic taxa richness and diversity at the perennial headwater site were significantly greater than richness and diversity at intermittent sites, whereas the downstream perennial site did not differ significantly from two of the intermittent sites. The larger magnitude of floods at the downstream perennial site resulted in greater losses in richness than at the upstream intermittent and perennial sites. Both classification and ordination revealed that benthic assemblage composition was more strongly correlated with time since last disturbance and season than with static hydrologic descriptors, microhabitat measures, or assemblage characteristics (richness or density). Richness of aerial colonization and drift samples collected at intermittent sites was greatest at the site with the nearest upstream perennial surface water. Because Kings Creek is a relatively pristine stream, these data may be useful as a baseline for comparison with future efforts for bioassessment of intermittent prairie streams.