In an attempt to isolate the ascomycete Cryphonectria parasitica (Diaporthales, Valsaceae) from dead chestnut stems, we obtained three C. radicalis strains. All three strains were isolated in areas of Switzerland with high chestnut blight incidence. To confirm our species designation, we compared the three C. radicalis strains to hypovirus (hv)-free and hv-infected C. parasitica strains. The comparison revealed several distinctive characteristics. On potato dextrose agar in the dark, the C. radicalis strains produced a fluffy mycelium and small droplets of a purple exudate giving the mycelium a light pinkish appearance. On corn meal medium in the dark, the C. radicalis strains caused a color change of the medium to purple, whereas the C. parasitica strains did not cause any color change. Ascospores from C. radicalis were significantly smaller than C. parasitica ascospores and their dimensions fit within other published size ranges. Southern hybridization analysis of the two species using nuclear and mitochondrial probes support their taxonomic separation. This separation is further supported by the lack of successful interspecific crosses. In virulence tests on chestnut trees, the C. radicalis strains exhibited very low virulence, comparable to highly hypovirulent hv-infected C. parasitica strains. Our results suggest that C. radicalis still coexists with C. parasitica although at a low frequency.