Colletotrichum species are widely known as key anthracnose pathogens of several economic plants. In this study, Colletotrichum species associated with leaf anthracnose isolated from various plants in Thailand were subjected to morphological and molecular analyses. The ITS rDNA regions of these strains were sequenced and aligned with those of type strains in the genus in order to establish if they can be assigned to any known species. Strains that could not be identified were further sequenced for partial actin (ACT), β-tubulin (TUB2) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) genes and employed in a phylogenetic analysis to reveal their relationships with other closely related taxa. The multilocus sequence analysis, together with a critical examination of the phenotypic characters, revealed three new species. These are introduced as C. brevisporum, C. tropicicola and C. thailandicum and formally described, illustrated and compared with similar taxa.