Ecological studies of fungal communities increasingly rely on large scale sequencing of environmental DNA, followed by clustering of sequences into operational taxonomic units (OTUs). To date there is no objective method of clustering sequences into groups that is grounded in an evolutionary theory of what constitutes a biological lineage. Here I describe an approach developed by insect systematists that distinguishes population-level processes within lineages from processes associated with speciation and extinction, thus identifying a distinct point where extant lineages became independent, and use this approach to estimate diversity of fungal endophytes from surveys of environmental DNA. Compared to OTU-based approaches defined by fixed levels of sequence similarity, groups delineated by the evolutionarily-informed approach better explained variation in the distribution of fungi in relation to putative niche-based variables associated with host species identity, environmental variables, and aspects of how the sampled ecosystems were managed. These results suggest the evolutionarily-informed approach successfully groups environmental sequences of fungi into clusters that are ecologically more meaningful than more arbitrary approaches for estimating fungal diversity.