DNA barcoding is a commonly employed method for assessing fungal diversity and generating sequence data to understand species relationships. However, most groups of fungi require in-depth analysis of multilocus data to infer phylogeny and delimit species. I led development of a sequencing method for rapid and accurate targeted sequencing of hundreds of fungal specimens using the PacBio long-read sequencing platform. This method generates multilocus data from both nuclear ribosomal and single-copy loci, including haplotype data and potential pseudogenes, and is compatible with fresh, dried, and cultured specimens. Using this method, I found unexpectedly high levels of intraspecific and intraindividual genetic diversity within several fungal lineages, including groups with multi-nucleate cells which have impeded resolution of species concepts.