Cryphonectria hypovirus 1 (CHV-1) acts as a naturally occurring biological control agent for chestnut blight, a destructive fungal disease of chestnut trees, which has been introduced into Europe in the 1930s.We have determined partial nucleotide and deduced amino acid sequences of the ORF A of 47 CHV-1 isolates collected in Europe over a period of 28 years. Phylogenetic analysis revealed the presence of four groups or single viruses, which showed sequence divergences ranging from 11 to 19%. These results confirm the previous subtype classification based on RFLP markers, with the exception of the two CHV-1 subtypes E and D, which appear to be related closer than anticipated previously. Dates of divergences between CHV-1 subtypes, calculated from nucleotide substitution rates, indicate that the CHV-1 subtypes diverged several hundreds years ago. Our results suggest that the genetic variation among CHV-1 subtypes did not evolve in Europe and support the hypothesis of multiple introductions of CHV-1 into Europe.