ABSTRACT Ninety-five isolates of Colletotrichum including 81 isolates of C. acutatum (62 from strawberry) and 14 isolates of C. gloeosporioides (13 from strawberry) were characterized by various molecular methods and pathogenicity tests. Results based on random amplified polymorphic DNA (RAPD) polymorphism and internal transcribed spacer (ITS) 2 sequence data provided clear genetic evidence of two subgroups in C. acutatum. The first subgroup, characterized as CA-clonal, included only isolates from strawberry and exhibited identical RAPD patterns and nearly identical ITS2 sequence analysis. A larger genetic group, CA-variable, included isolates from various hosts and exhibited variable RAPD patterns and divergent ITS2 sequence analysis. Within the C. acutatum population isolated from strawberry, the CA-clonal group is prevalent in Europe (54 isolates of 62). A subset of European C. acutatum isolates isolated from strawberry and representing the CA-clonal and CA-variable groups was assigned to two pathogenicity groups. No correlation could be drawn between genetic and pathogenicity groups. On the basis of molecular data, it is proposed that the CA-clonal subgroup contains closely related, highly virulent C. acutatum isolates that may have developed host specialization to strawberry. C. gloeosporioides isolates from Europe, which were rarely observed were either slightly or nonpathogenic on strawberry. The absence of correlation between genetic polymorphism and geographical origin in Colletotrichum spp. suggests a worldwide dissemination of isolates, probably through international plant exchanges.