Abstract Restriction fragment length polymorphisms (RFLPs) of the ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of isolates of the strawberry anthracnose pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides were analysed using rDNA from Saccharomyces carlsbergensis and mtDNA extracted from C. acutatum, C. fragariae and C. gloeosporioides as probes. These analyses revealed considerable heterogeneity within C. acutatum from diverse hosts. The European strawberry isolates formed a discrete group while the American strawberry isolates fell into a broad group which included isolates from other hosts. No polymorphisms in either rDNA or mtDNA were observed among C. gloeosporioides isolates from strawberry. C. fragariae isolates divided into two groups with distinct rDNA and mtDNA patterns. Random amplified polymorphic DNA (RAPD) analysis grouped isolates in a similar manner to the mtDNA RFLP analysis. From sequencing data, the internally transcribed spacer (ITS) 1 region of the rDNA repeat unit of C. gloeosporioides, C. fragariae and C. acutatum was shown to be 171, 171 or 172 and 180 or 181 bases, respectively. C. gloeosporioides and C. fragariae differed from one another by only three to seven bases compared with C. acutatum which differed from C. gloeosporioides and C. fragariae by approx. 36–37 bases. ITS 1 data for C. acutatum generally support RFLP and RAPD taxonomies; differences of eight-eleven bases between European strawberry isolates and all other isolates studied being the greatest.