Tallgrass prairie streams and their fauna are understudied, due in part to their rarity. The composition of prairie stream communities differs geographically due to regionally and locally determined environmental characteristics and varies over time due to disturbance events. This research focused on expanding the current knowledge of temporal and spatial variation in the macroinvertebrate communities of Missouri prairie streams. Intra-seasonal variation in the macroinvertebrate community of one prairie stream was correlated with changes in the stream environment and resulted in three relatively short-lived but significant community groupings. These intra-seasonal community shifts also affected functional feeding group structure and led to significant differences in biomonitoring measures over the course of weeks. For situations in which small, dynamic stream systems need to be monitored, sampling should be conducted at frequent intervals to capture changes in communities that may occur within seasons. The regional diversity and community structure of prairie headwater stream communities is in part driven by both the intermittent nature and the relative scarcity of prairie stream habitat. The macroinvertebrate communities of the headwater networks from five prairies in Missouri were unique. Differences in their taxonomic composition led to differences in biomonitoring metric values. The distinct community compositions of the headwater prairie streams studied and the high regional diversity documented are likely driven by geographic isolation rather than local environmental factors. Each of these prairie stream systems is an important contributor to species diversity at a regional level. Conservation and restoration efforts must work to preserve the biotic diversity in prairie streams to support existing macroinvertebrate communities that remain. For lotic insects to maintain stable populations across fragmented prairie patches and their intermittent streams in Missouri, dispersal must occur between patches to enable recolonization of streams after disturbance. Leptophlebia konza Burian was thought to be restricted to its type locality of Konza Prairie, Kansas; however, a population was discovered at Hi Lonesome Prairie in Missouri during an extensive search of 54 prairies. DNA sequence data from cytochrome oxidase subunit 1, CO1, show that both populations of L. konza lost genetic diversity during a recent bottleneck, either because of habitat fragmentation or competition from other species, including Leptophlebia johnsoni McDunnough. The rarity of L. konza populations across Missouri when taken into consideration with DNA sequence data suggest that L. konza populations are unstable and unable to disperse between remaining prairie patches. Water quality monitoring and research activities taking place in prairie streams must take into account spatial and temporal variation in both community structure and genetic diversity across these landscapes.