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During a study of <i>Botryosphaeriaceae</i> species associated with grapevine trunk diseases in the Czech Republic, a collection of 22 <i>Botryosphaeriaceae</i>-like strains were isolated from four cultivars (Blaufränkisch, Pálava, Pinot Noir, and Welschriesling) in four distinct vineyards. Based on morphology and DNA sequence data (ITS, <i>tub2</i>, and <i>tef</i>), four species were identified: <i>Botryosphaeria dothidea</i>, <i>Diplodia mutila</i>, <i>D. seriata</i>, and <i>Neofusicoccum parvum</i>. These species are reported for the first time from grapevine in the Czech Republic. Relationships between vascular lesions and particular species were highlighted in this study. <i>Diplodia seriata</i> was the most frequently isolated species, present in all four sampled cultivars, while <i>D. mutila</i> was the least frequent, present only in ‘Pálava’. The cultivar Pinot Noir was the most tolerant host for <i>Botryosphaeriaceae</i> fungi.