Grapevine Trunk Diseases (GTDs) cause important economic losses in vineyards. Diagnosis of pathogens causing GTDs is usually achieved using culture methods from symptomatic plants, but these methods may not be definitive. This study aimed to detect three fungi associated with GTDs in Chile (Diplodia seriata, Phaeomoniella chlamydospora and Eutypa lata) using qPCR methods, after extracting DNA from asymptomatic woody pruning material taken from 912 grapevine plants and 346 rootstocks. Pathogen incidence and distribution across different regions, vineyards, grape and wine cultivars, plant ages and rootstocks were determined. Forty percent of assessed grapevine plants and 42% of rootstocks were positive for the assayed GTD pathogens. The fungus with the greatest incidence was D. seriata (36% of plants and 30% of rootstocks), followed by E. lata (6% of plants and 20% of rootstocks) and P. chlamydospora (4% of plants and 5% of rootstocks). Positive relationships were detected between the presence of D. seriata and P. chlamydospora and grapevine age. However, the three fungi were randomly distributed across graprevine plants and rootstocks since none of the assessed factors had statistically significant influence in their distribution. This study is the first to assess incidence and distribution of E. lata in Chile. The qPCR-based method described could be used for detecting fungi in asymptomatic nursery plants, and be used to avoid distribution of infected plants into new vineyard plantings.