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Plant-associated fungi can ameliorate abiotic stress in their hosts, and changes in these fungal communities can alter plant productivity, species interactions, community structure and ecosystem processes. We investigated the response of root-associated fungi to experimental drought (66% reduction in growing season precipitation) across six North American grassland ecosystem types to determine how extreme drought alters root-associated fungi, and understand what abiotic factors influence root fungal community composition across grassland ecosystems. Next generation sequencing of the fungal ITS2 region demonstrated that drought primarily re-ordered fungal species’ relative abundances within host plant species, with different fungal responses depending on host identity. Grass species that declined more under drought trended toward less community re-ordering of root fungi than species less sensitive to drought. Host identity and grassland ecosystem type defined the magnitude of drought effects on community composition, diversity, and root colonization, and the most important factor affecting fungal composition was plant species identity.