The causal agent of ash dieback, the hyphomycete Chalara fraxinea, was isolated from a Fraxinus excelsior cv. Pendula tree, in the Arboretum Křtiny between Křtiny and Jedovnice in Drahany Highland, and subsequently from several other locations in South Moravia in the Czech Republic. The infection was associated with severe twig dieback and symptoms of ash dieback were observed in many locations across the Czech Republic. The morphology of C. fraxinea is described and an ITS sequence is provided. Hymenoscyphus albidus, the teleomorph of C. fraxinea, is known as a quit common species from precedent mycofloristic studies provided in different areas in the Czech Republic.