Grapevine trunk diseases (GTDs) are a threat to grape production worldwide, with a diverse collection of fungal species implicated in disease onset. Due to the long-term and complex nature of GTDs, simultaneous detection of multiple microbial species can enhance understanding of disease development. We used DNA metabarcoding of ribosomal internal transcribed spacer 1 (ITS1) sequences, supported by specific PCR and microbial isolation, to establish the presence of trunk pathogens across 11 vineyards (11-26 years old) over three years in Marlborough, the largest wine producing region in New Zealand. Using a reference database of trunk pathogen sequences, species previously associated with GTD, such as Cadophora luteo-olivacea, Diplodia seriata, Diplodia mutila, Neofusicoccum australe, and Seimatosporium vitis, were identified as highly represented across the vineyard region. The well-known pathogens Phaeomoniella chlamydospora and Eutypa lata had especially high relative abundance across the dataset, with P. chlamydospora reads present between 22 and 84% (average 52%) across the vineyards. Screening of sequences against broader, publicly available databases revealed further fungal species within families and orders known to contain pathogens, many of which appeared to be endemic to New Zealand. The presence of several wood-rotting basidiomycetes (mostly Hymenochaetales) was detected for the first time in the Marlborough vineyard region, notably, the native Inonotus nothofagii which was present at 1-2% relative abundance in two vineyards., (© 2022. The Author(s).)