Genetic divergence among 57 isolates of Colletotrichum lindemuthianum collected in north, central and south America and The Netherlands was assessed on the basis of PCR-RFLP analysis and sequencing of the nuclear rDNA region of the two internal transcribed spacers (ITS 1 and ITS 2) and the 5·8S rRNA gene. A reproducible 0·58 kb fragment was amplified in all 57 isolates. Races of C. lindemuthianum formed two groups based on the restriction of the PCR-amplified ITS regions. Group I consisted mainly of Middle American races (65%), whereas 85% of Andean races belonged in group II. Genetic distances calculated from the sequence polymorphism in the rDNA region ranged from 0·2 to 1·8% among 14 isolates of C. lindemuthianum. The neighbour-joining and parsimony analyses of the sequence data showed no association of any particular ITS genotype with host gene pool, virulence or geographic origin of races. These data suggest that virulence can arise in different geographic areas at different times, independent of genetic background. Molecular polymorphism among isolates of races 7, 17, 31 and 73 collected in different countries was demonstrated by RFLP analysis of the ITS regions. A similar lack of agreement was observed in the sequence data between isolates of race 73 from Mexico and the United States and the Jukes–Cantor genetic distance between the isolates was large (0·9%). These findings support a level of molecular variability within C. lindemuthianum greater than the variability previously characterized by virulence analysis and suggest independent evolution of specific virulence patterns.