Cryphonectria hypovirus 1 (CHV-1) is responsible for hypovirulence (reduced virulence) in the chestnut blight fungus Cryphonectria parasitica and has been used as a biocontrol agent against this introduced tree pathogen. However, the development of self-sustaining hypovirulence following the deployment of virus strains has never clearly been demonstrated. We assessed the prevalence of subtypes of CHV-1 in southeastern France, where a specific CHV-1 subtype (F1) has been released for biological control at several chestnut sites. Using these data, we explored the potential effects of control treatments, fungal vegetative incompatibility and the ecological fitness of viral subtypes on the establishment of CHV-1 in C. parasitica populations. The prevalence of CHV-1 in C. parasitica cankers varied from 31 to 90% between sites and was not related to biological control treatment or to the diversity of vegetative compatibility types of the fungal population. Our study revealed that the CHV-1 subtype F1 used for biological control did not become established in the study area. Of the 79 hypoviruses identified, 78 belonged to CHV-1 subtype I, and one to subtype F2. Fungal isolates infected with CHV-1 subtypes F1 or F2 grew significantly slower and had lower levels of sporulation than isolates infected with the naturally occurring subtype I viruses. The greater ecological fitness of subtype I than of subtypes F1 and F2 probably accounts for its greater invasiveness. Our results indicate that different CHV-1 subtypes could be used differently in the biological control of chestnut blight.