Grapevine trunk diseases (GTD) are one of the most important constraints for grapevine and wine production worldwide. Most of the published studies on these diseases in Europe relate to the major vine growing regions mainly located in southern Europe. This is the first study of GTD incidence and associated fungal pathogens in Czech vineyards, as an example of the north-eastern European grape-growing region. Annual incidence of GTD symptoms in vineyards of cvs. ‘Chardonnay’ and ‘Cabernet Sauvignon’ in three Czech locations was surveyed. On average, 3.7% of the ‘Chardonnay’ plants showed GTD symptoms and 0.7% suddenly collapsed each year. Within the ‘Cabernet Sauvignon’ vineyards, 10.1% of the plants showed GTD symptoms and 2.5% suddenly collapsed. Several fungal pathogens were associated with the suddenly collapsed grapevines, and the most frequent was Phaeomoniella chlamydospora (77% of analyzed grapevines), followed by Eutypa lata (62%), Diaporthe ampelina (31%), Botryosphaeria dothidea (15%) and Fomitiporia mediterranea (15%).