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Colletotrichum is among the most important genera of fungal plant pathogens. Molecular phylogenetic studies over the last decade have resulted in a much better understanding of the evolutionary relationships and species boundaries within the genus. There are now approximately 200 species accepted, most of which are distributed among 13 species complexes. Given their prominence on agricultural crops around the world, rapid identification of a large collection of Colletotrichum isolates is routinely needed by plant pathologists, regulatory officials, and fungal biologists. However, there is no agreement on the best molecular markers to discriminate species in each species complex. Here we calculate the barcode gap distance and intra/inter-specific distance overlap to evaluate each of the most commonly applied molecular markers for their utility as a barcode for species identification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone-3 (HIS3), DNA lyase (APN2), intergenic spacer between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), and intergenic spacer between GAPDH and a hypothetical protein (GAP2-IGS) have the properties of good barcodes, whereas sequences of actin (ACT), chitin synthase (CHS-1) and nuclear rDNA internal transcribed spacers (nrITS) are not able to distinguish most species. Finally, we assessed the utility of these markers for phylogenetic studies using phylogenetic informativeness profiling, the genealogical sorting index (GSI), and Bayesian concordance analyses (BCA). Although GAPDH, HIS3 and β-tubulin (TUB2) were frequently among the best markers, there was not a single set of markers that were best for all species complexes. Eliminating markers with low phylogenetic signal tends to decrease uncertainty in the topology, regardless of species complex, and leads to a larger proportion of markers that support each lineage in the Bayesian concordance analyses. Finally, we reconstruct the phylogeny of each species complex using a minimal set of phylogenetic markers with the strongest phylogenetic signal and find the majority of species are strongly supported as monophyletic., (Copyright © 2019 Elsevier Inc. All rights reserved.)