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Different distance-based threshold selection approaches were used to assess and compare use of the internal transcribed spacer (ITS) region to distinguish among 901 Cortinarius species represented by >3000 collections. Sources of error associated with genetic markers and selection approaches were explored and evaluated using MOTUs from genus and lineage based-alignments. Our study indicates that 1%-2% more species can be distinguished by using the full-length ITS barcode as compared to either the ITS1 or ITS2 regions alone. Optimal threshold values for different picking approaches and genetic marker lengths inferred from a subset of species containing major lineages ranged from 97.0% to 99.5% sequence similarity using clustering optimization and UNITE SH, and from 1% to 2% sequence dissimilarity with CROP. Errors for the optimal cutoff ranged from 0% to 70%, and these can be reduced to a maximum of 22% when excluding species lacking a barcode gap. A threshold value of 99% is suitable for distinguishing species in the majority of lineages in the genus using the entire ITS region but only 90% of the species could be identified using just the ITS1 or ITS2 region. Prior identification of species, lacking barcode gaps and their subsequent separate analyses, maximized the accuracy of threshold approaches. [ABSTRACT FROM AUTHOR]