Strains of Colletotrichum species derived from diseased strawberry plants from a wide geographical range were studied using mitochondrial and ribosomal DNA RFLPs, and acetyl and propionyl esterase isoenzymes. Two major species aggregates were detected, centred on C. acutatum and C. gloeosporioides respectively, with significant further subdivision. There were apparent discrepancies in the hierarchical nesting of some taxon groups based on the different molecular techniques. Strains assigned to C. acutatum fell into several rDNA RFLP groups, but there was less variation in mtDNA RFLP band patterns. There appears to be at least one probably clonal population in the U.S.A. which is also present in Europe, and a less well-defined series of groups which are at least sometimes sexually reproducing. Strains assigned to C. fragariae were found not to have distinct rDNA band patterns from the teleomorph linked strains studied, which had been referred to as C. gloeosporioides. They did vary in this respect from C. gloeosporioides associated with Citrus, its type host. It was, therefore, concluded that all the strains studied with cylindrical conidia should be placed within C. fragariae, which is confirmed as separate from C. gloeosporioides and recognized as a holomorphic taxon. Nevertheless, a separate asexually reproducing infraspecific group was distinguishable using mtDNA information. An epitype is designated for C. fragariae.