The grapevine trunk disease, Eutypa dieback (ED), causes significant vine decline and yield reduction. For many years, the fungusEutypa latawas considered the main pathogen causing ED of grapevines in Australia. Recent studies showed other Diatrypaceous fungi were also associated with vines exhibiting dieback symptoms but there is limited information on how these fungal pathogens spread in vineyards. Thus, information on the spore dispersal patterns of Diatrypaceous fungi in different wine regions will assist in identifying high-risk infection periods in vineyards. Using more than 6800 DNA samples from airborne spores collected from eight wine regions in south-eastern Australia over 8 years using a Burkard spore trap, this study investigated the diversity and abundance of Diatrypaceous species, using multi-faceted molecular tools. A multi-target quantitative PCR (qPCR) assay successfully detected and quantified Diatrypaceous spores from 30% of the total samples with spore numbers and frequency of detection varying between regions and years. The high-resolution melting analysis (HRMA) coupled with DNA sequencing identified seven species, withE.latabeing present in seven regions and the most prevalent species in the Adelaide Hills, Barossa Valley and McLaren Vale.Cryptovalsa ampelinaandDiatrype stigmawere the predominant species in the Clare Valley and Coonawarra, respectively whileEutypella citricolaandEu.microthecadominated in the Hunter Valley and the Riverina regions. This study represents the first report ofD.stigmaandCryptosphaeria multicontinentalisin Australian vineyards. This study further showed rainfall as a primary factor that triggers spore release, however, other weather factors that may influence the spore release in different climatic regions of Australia still requires further investigation.