Technical Abstract: Colletotrichum acutatum J. H. Simmonds and C. gloeosporioides (Penz.) Penz. & Sacc. in Penz. [teleomorph Glomerella cingulata (Stoneman) Spauld. & H. Schrenk] are important pathogens of strawberry and other crops. In this study, 47 isolates representing C. acutatum (36 isolates), C. gloeosporioides (9 isolates), and C. fragariae (2 isolates) from 10 fruit crops grown in the southeastern United States were selected for genotypic and phenotypic analysis. Isolate identification was based on conidial shape, colony color, and growth rate at 25oC. The pathogen genotype was described by random amplified polymorphic DNAs (RAPDs), and the phenotype was represented by conidial shape, growth radials on potato dextrose agar (PDA) at 22 and 30oC for 7 days, and sensitivity to benomyl. Seven RAPD groups were identified. RAPD Group 4 contained C. gloeosporioides isolates characterized by rounded-end conidia, high sensitivity to benomyl, and larger growth radials on PDA at 22 and 30oC. RAPD group 7 included only the two C. fragariae isolates characterized by a conidial shape of one end tapered and the other end rounded, and larger growth radials on PDA at 22 and 30oC. RAPD groups 1, 2, 3, 5 and 6 were composed of isolates of C. acutatum typical for their conidial morphology of both ends acutely tapered, smaller growth radials on PDA, and low benomyl sensitivity. All four C. acutatum isolates from strawberry were included in RAPD group 5. Comparison of growth radials at 22 and 30oC revealed the presence of a warm temperature cluster of C. acutatum represented by isolates in RAPD group 2, with a larger growth radial at 30oC (51 mm) than that at 22oC (27 mm). Most isolates in the other C. acutatum RAPD groups grew less at 30oC than at 22oC. RAPD group 6 was composed of only the two C. acutatum isolates from citrus.