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Background and Aims: Botryosphaeriaceae species are recognised as important pathogens of grapevines both in Australia and overseas. The identity, prevalence and distribution of Botryosphaeriaceae species in vineyards throughout the major winegrowing regions of New South Wales (NSW) and South Australia (SA) was determined to provide a foundation for improved disease prevention and management. Methods and Results: Field surveys from 91 vineyards across NSW and SA resulted in the collection of 2239 diseased wood samples and subsequent isolation of 1258 Botryosphaeriaceae isolates. Morphological identification along with phylogenetic analysis of ribosomal DNA internal transcribed spacer regions (ITS1-5.8S-ITS2) and partial sequences of the translation elongation factor 1-α gene (EF1-α) showed that eight Botryosphaeriaceae species from four phylogenetic lineages occur on grapevines in eastern Australia, including Diplodia seriata, Diplodia mutila, Lasiodiplodia theobromae, Neofusicoccum parvum, Neofusicoccum australe, Botryosphaeria dothidea, Dothiorella viticola and Dothiorella iberica. Conclusions: The prevalence of individual species varied according to geography and climate. Species of Diplodia and Dothiorella, characterised by thick-walled, pigmented conidia were the most prevalent and were distributed widely throughout both NSW and SA. Species with hyaline conidia, such as Neofusicoccum and Fusicoccum, were isolated less frequently and displayed more limited geographic ranges, whilst only a single isolate of Lasiodiplodia was recovered, this being from the northern most region of NSW. Significance of the Study: The identification of eight taxa within the Botryosphaeriaceae, and their distributions throughout south-eastern Australia was established and discussed in context with climate, reported optimum growth temperatures, and more recent taxonomic and nomenclatural revisions. We established a sound base for control strategies based on the prevailing species in Australian viticultural regions. [ABSTRACT FROM AUTHOR]