A large number of species in the genus <i>Colletotrichum</i> have been reported as causal agents of anthracnose on crops and wild plants in Korea. Many <i>Colletotrichum</i> isolates from the country preserved in the Korean Agricultural Culture Collection (KACC) were previously identified based on host plants and morphological characteristics, and it may lead to species misidentification. Thus, accurate fungal species identification using multilocus sequence analyses is essential for understanding disease epidemiology and disease management strategies. In this study, combined DNA sequence analyses of internal transcribed spacer, <i>gapdh</i>, <i>chs-1</i>, <i>his3</i>, <i>act</i>, <i>tub2</i>, and <i>gs</i> were applied to re-identify 27 <i>Colletotrichum</i> isolates in KACC. The phylogenetic analyses showed that the isolates resulted in 11 known species, they belong to the <i>C. dematium</i> species complex (<i>C. hemerocallidis</i>, <i>C. jinshuiense</i>, and <i>C. spinaciae</i>), the <i>C. magnum</i> complex (<i>C. kaifengense</i> and <i>C</i>. cf. <i>ovatense</i>), the <i>C. orchidearum</i> complex (<i>C. cattleyicola</i>, <i>C. plurivorum</i>, <i>C. reniforme</i>, and <i>C. sojae</i>) and the <i>C. orbiculare</i> complex (<i>C. malvarum</i> and <i>C. orbiculare</i>). Of them, <i>C. cattleyicola</i>, <i>C. hemerocallidis</i>, <i>C. kaifengense</i>, and <i>C. reniforme</i> were unrecorded species in Korea. In the view of host-fungus combinations, 10 combinations are newly reported in the world and 12 are new reports in Korea, although their pathogenicity on the host was not confirmed.