Grapevine trunk diseases are regarded as one of the most important threats to the sustainability of the Australian wine industry (Sosnowski et al., 2017; 2022). Eutypa dieback is a fungal disease included in the grapevine trunk disease complex and is caused mainly by the fungus Eutypa lata, but other Diatrypaceae fungi may be involved (Trouillas et al., 2010; 2011). Cryptovalsa ampelina is one of the fungal species implicated in this disease complex, however little is known about this fungus despite it being prevalent in some Australian wine regions (Billones-Baaijens et al., 2023). To further understand the presence of C. ampelina in Australian vineyards, woody tissues from grapevines containing dead wood were sampled from the following winegrape growing regions, Tumbarumba, Griffith and Orange in NSW; Clare Valley, Barossa Valley, Adelaide Hills, McLaren Vale and Riverland in South Australia; Coal, Huon and Derwent River Valleys in Tasmania and Margaret River in Western Australia. Grapevines that were 25 years old or older (cvs. Shiraz, Cabernet Sauvignon, Pinot Noir, Sauvignon Blanc and Chardonnay) were sampled for the presence of C. ampelina using two different methods. Internal grapevine tissues were extracted with a drill, and wood shavings were plated on potato dextrose agar with chloramphenicol (PDA-C) to isolate the fungus. Additionally, the external wood was examined under a dissecting microscope to identify dead stromata containing perithecia. Perithecia were inspected under a compound microscope, and when asci contained 32 spores (characteristic of C. ampelina), these were cultured on PDA-C. C. ampelina was identified morphologically, and the mycelium was sub-cultured onto fresh PDA. The identity of C. ampelina was also confirmed using Loop-mediated isothermal amplification (LAMP) specific primers (Sosnowski et al., 2022) and by DNA sequencing. In total, 82 blocks from 43 vineyards located in 12 wine regions were surveyed and 36 isolates of C. ampelina were recovered. C. ampelina was found to be prevalent in the Clare and Derwent Valley regions and was detected in all vineyards sampled from these regions. It was detected from all cultivars sampled, however it is unknown if cultivars differ in susceptibility. These results provide new information on the distribution of this pathogen in Australian vineyards. Subsequent experiments will be established to assess the pathogenicity and optimum climatic conditions for infection of grapevines by C. ampelina.