The Colletotrichum gloeosporioides species complex is defined genetically based on a strongly supported clade within the ITS gene tree. Twenty-two species and one subspecies are accepted within this complex, including C. asianum, C. cordylinicola, C. fructicola, C. gloeosporioides, C. horii, C. kahawae subsp. kahawae, C. musae, C. nupharicola, C. psidii, C. siamense, C. theobromicola, C. tropicale, and C. xanthorrhoeae, along with newly described species such as C. aenigma, C. aeschynomenes, C. alatae, C. alienum, C. aotearoa, C. clidemiae, C. kahawae subsp. cigaro, C. salsolae, and C. ti, and the nom. nov. C. queenslandicum (for C. gloeosporioides var. minus). All taxa are defined genetically using multi-gene phylogenies. Morphological descriptions are provided for species without modern descriptions. ITS sequences, the official barcoding gene for fungi, are insufficient to reliably distinguish many species, particularly those in the Musae clade and Kahawae clade. Single genes or combinations of genes, such as glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase, are used for reliable species-level identification. The study clarifies genetic and taxonomic relationships within the complex using a wide range of isolates. The complex includes species with broad host and geographic ranges, such as C. fructicola, C. kahawae subsp. cigaro, C. siamense, and C. theobromicola. The study also addresses the taxonomic status of several species and subspecies, including the new subspecies C. kahawae subsp. cigaro. The results show that the complex consists of two main clades, the Musae clade and the Kahawae clade, with several species phylogenetically well supported. The study provides genetic and morphological data for the accepted species and discusses the implications for species identification and biosecurity. The study also highlights the importance of using multiple genes for accurate species identification, as many of these fungi are of biosecurity significance. The study includes detailed descriptions of the new species and subspecies, as well as their geographic distribution and host range. The study also discusses the taxonomic status of several species and subspecies, including the new subspecies C. kahawae subsp. cigaro. The study provides a comprehensive overview of the genetic and morphological diversity within the Colletotrichum gloeosporioides species complex.The Colletotrichum gloeosporioides species complex is defined genetically based on a strongly supported clade within the ITS gene tree. Twenty-two species and one subspecies are accepted within this complex, including C. asianum, C. cordylinicola, C. fructicola, C. gloeosporioides, C. horii, C. kahawae subsp. kahawae, C. musae, C. nupharicola, C. psidii, C. siamense, C. theobromicola, C. tropicale, and C. xanthorrhoeae, along with newly described species such as C. aenigma, C. aeschynomenes, C. alatae, C. alienum, C. aotearoa, C. clidemiae, C. kahawae subsp. cigaro, C. salsolae, and C. ti, and the nom. nov. C. queenslandicum (for C. gloeosporioides var. minus). All taxa are defined genetically using multi-gene phylogenies. Morphological descriptions are provided for species without modern descriptions. ITS sequences, the official barcoding gene for fungi, are insufficient to reliably distinguish many species, particularly those in the Musae clade and Kahawae clade. Single genes or combinations of genes, such as glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase, are used for reliable species-level identification. The study clarifies genetic and taxonomic relationships within the complex using a wide range of isolates. The complex includes species with broad host and geographic ranges, such as C. fructicola, C. kahawae subsp. cigaro, C. siamense, and C. theobromicola. The study also addresses the taxonomic status of several species and subspecies, including the new subspecies C. kahawae subsp. cigaro. The results show that the complex consists of two main clades, the Musae clade and the Kahawae clade, with several species phylogenetically well supported. The study provides genetic and morphological data for the accepted species and discusses the implications for species identification and biosecurity. The study also highlights the importance of using multiple genes for accurate species identification, as many of these fungi are of biosecurity significance. The study includes detailed descriptions of the new species and subspecies, as well as their geographic distribution and host range. The study also discusses the taxonomic status of several species and subspecies, including the new subspecies C. kahawae subsp. cigaro. The study provides a comprehensive overview of the genetic and morphological diversity within the Colletotrichum gloeosporioides species complex.