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<div><i>Populus </i>is emerging as an important model tree genetic system for studies aimed at understanding forest ecosystems and the interactions of these with the associated microbiota. <i>Populus</i> species are known to establish associations with arbuscular and ectomycorrhizal fungi which vary depending on the host species. Recent studies have unlocked a great diversity of fungal endophytes in <i>Populus, </i>associated with different plant tissues. The ecological and physiological roles of fungal endophytes in the <i>Populus</i>-microbe interaction have not been fully characterized. We describe a novel approach using multilocus sequence typing (MLST) approach using PacBio and the downstream analysis to genotype individuals in single sequencing run, providing enough loci to characterize a species complex. This approach was applied to <i>Ilyonectria</i> complex, an endophyte present in cottonwood (<i>Populus trichocarpa </i>and <i>Populus deltoides</i>), but also associated with black-foot disease on grapevines. The methodology could be applied to study the phylogenetics and systematics of other fungal groups. In addition, genomes of <i>Populus</i> root-associated fungal endophytes are being sequenced. The data extracted from these genomes provides insight into the lifestyle strategies of various endophyte species, and provides a framework for metatranscriptomic approaches to understand the role of endophytic fungi.</div>