Colletotrichum acutatum J. H. Simmonds is an important fungal pathogen that causes anthracnose disease in crops. In this study, 59 C. acutatum isolates from regions in South Korea were collected and phenotypically compared. The average colony diameters of isolates from Gyeongsangbuk-do, Chungcheongbuk-do, and Gangwon-do were 4.36 cm, 4.24 cm, and 4.38 cm, respectively, following 6 days of growth on V8 juice agar. The average conidium concentrations of the isolates from each province were 14.43, 12.83, and 10.17 × 104/mL after 7 days of growth on V8A. Amplified fragment length polymorphism(AFLP) analysis using three primer pairs identified 5 polymorphic bands and 56 monomorphic bands. Two polymorphic bands were identified using the E-ACC/M-CTT primer pair, one polymorphic band was identified using the E-ACG/M-CTG primer pair, and two polymorphic bands were identified using the E-ACG/M-CTT primer pair. A dendrogram was constructed using the AFLP data, and four clusters were identified.