In disease ecology, parasite transmission is a key parameter important at both epidemiological and evolutionary scales. Mycoviruses can be transmitted both horizontally and vertically. Their horizontal transmission is strongly restricted by the host vegetative compatibility system, which controls the outcome of somatic fusion in fungi, and by the same way, may limit mycovirus transmission. However, most of current knowledge and predictive capabilities regarding these host/pathogen systems are derived from studies pairing fungal mycelia on artificial medium. An original bioassay method, using infected mycelia as well as asexual spores, had been developed to assess in situ transmission of Cryphonectria Hypovirus-1 (CHV1), a mycovirus of Cryphonectria parasitica that causes chestnut blight. For every pair of different vegetative compatibility types tested, rates of CHV1 transmission were always superior in situ than in vitro. This study supports the hypothesis that the natural ability of CHV1 to migrate within a fungal population composed of different vegetative compatible types may have been underestimated by in vitro essays. This result offers opportunities for a biological control of fungal diseases with mycoviruses.