Bitter rot caused by Colletotrichum species is a common fruit rotting disease of apple and one of the economically important disease in worldwide. In 2015 and 2016, distinct symptoms of bitter rot disease were observed in apple orchards in five regions of South Korea. In the present study, infected apples from these regions were utilized to obtain eighteen isolates of Colletotrichum spp. These isolates were identified and characterized according to their morphological characteristics and nucleotide sequence data of internal transcribed spacer regions and glyceraldehyde-3-phosphate-dehydrogenase. Molecular analyses suggested that the isolates of Colletotrichum causing the bitter rot disease in South Korea belong to 4 species: C. siamense; C. fructicola; C. fioriniae and C. nymphaeae. C. siamense and C. fructicola belonged to Musae Clade of C. gloeosporioides complex species while C. fioriniae and C. nymphaeae belonged to the Clade 3 and Clade 2 of C. acutatum complex species, respectively. Additionally, we also found that the isolates of C. gloeosporioides species-complex were more aggressive than those in the C. acutatum species complex via pathogenicity tests. Taken together, our results suggest that accurate identification of Colletotrichum spp. within each species complex is required for management of bitter rot disease on apple fruit in South Korea.