Chestnut rot is an important disease of chestnut trees in Oceania (Australia, New Zealand), and Europe (Italy, France, Switzerland, United Kingdom). The causal agent has been identified as the fungus Gnomoniopsis smithogilvyi ( Gnomoniaceae , Diaporthales ). Koch’s Postulates was demonstrated on nuts from three Australian chestnut varieties with G. smithogilvyi from Australia, New Zealand and Italy. The Australian and New Zealand isolates were pathogenic on all three varieties, however the Italian isolate produced smaller lesions on all three, and was only mildly pathogenic on Decoppi Marone. Infection of chestnut floral and vegetative tissues was investigated during the chestnut phenological stages of flowering (sampled twice over two years), immature nuts and burrs, mature nuts and burrs, and tree dormancy (immature nuts and burrs, mature nuts and burrs, and tree dormancy were sampled once in one year). Gnomoniopsis smithogilvyi was present in all the sampled phenological stages, most frequently from female flowers during the flowering period. In the first year, the fungus infected 82% of female flowers, while in the second year only 10% were infected. Ascospore trapping in a laboratory chamber experiment confirmed ascopores are released in to the air from infected burrs. Ascospore trapping in an Australian chestnut orchard during the flowering period also showed ascospores are released in to the air, with peak trapping times at sunset and the hours following sunset (8-11 pm), and the hours following sunrise (7-9 am). These experiments support the hypothesis that ascospores of G. smithogilvyi infect chestnut trees, particularly the flowers. The fungus then exists as a latent pathogen in reproductive and vegetative tissues, leading to the development of the disease during nut maturity.