Cryphonectria hypovirus 1 (CHV1) is a double-stranded (ds)RNA virus without a capsid that infects phytopathogenic fungus Cryphonectria parasitica, the causal agent of chestnut blight. This virus reduces virulence and reproductive capacities of the fungus and thus is very important in biological control of the chestnut blight. If a hypovirulent fungus carrying CHV1 is introduced into an active canker caused by virulent C. parasitica strain, canker expansion ceases and callus is formed. The aim of this research was to evaluate the prevalence of CHV1 in chestnut calli. A total of 24 calli from Ozalj and Markuševac were included in the research. Observed presence of virulent fungal strains in chestnut calli was high. As many as 29.16% of calli had only virulent fungal strains in their tissue, 45.84% of calli had the combination of virulent and hypovirulent fungal strains, and only 25% of calli had solely hypovirulent fungal strains in their tissue. It is assumed that this could have happened because of the spontaneous loss of virus from hypovirulent fungal strains or due to the loss of whole hypovirulent fungal strain and colonization of the callus by a new, virulent strain which could reinitiate the infection. In order to ascertain the reason for the loss of hypovirulence, it is necessary to monitor changes in the callus over a longer period of time.