Fungi play myriad roles in streams, including but not limited to aquatic hyphomycete fungi, which serve as the primary decomposers of plant litter and are an important food source for aquatic macroinvertebrates. Most research on aquatic fungi comes from perennial (continuously flowing) streams, but over half of the world’s streams are non-perennial (intermittent or ephemeral). Knowledge gaps about microbiome responses to stream drying make it difficult to predict the effects of climate change on freshwater ecosystem functions, as an increasing number of streams are undergoing flow intermittency. To build knowledge on intermittent stream mycobiomes in North America’s Great Plains, we used a community metabarcoding approach to examine fungal community composition across an intermittent stream network in the Konza Prairie, Kansas, USA. Environmental DNA (eDNA) was extracted from leaf litter, benthic sediments, epilithic biofilms, and surface water where present from 50 stream transects. Amplicon libraries were generated via metabarcoded PCR of the ITS1 locus, normalized and multiplexed, and sequenced via Illumina MiSeq. Resulting sequence reads were processed via the DADA2 pipeline in R to generate relative abundances of amplicon sequence variants (ASVs), with fungal taxonomy assigned using the UNITE database. Full factorial two-factor ANOVA showed that the effect of wet/dry on fungal ASV richness varied by substrate (F2,101=6.00, p=0.0035), with dry epilithon having double the richness as wet, while wet/dry had no effect on richness in litter or sediments. These preliminary results support the hypothesis that leaf litter and sediments serve as refugia, sheltering fungi from drying, while exposed rock surfaces experience higher community turnover. However, more detailed examination of fungal taxa and functional groups is needed to elucidate the impacts of flow intermittency on fungal community structure and function. Several genera of Verrucariaceae lichens were indicators of dry conditions in epilithon, supporting their use as biological indicators of hydrologic intermittency.