This study investigated the sources of inoculum of Botyosphaeriaceae species in three commercial grapevine nurseries in New Zealand. Botryosphaeriaceae propagules were detected on the surfaces of 33 to 100 % of canes and 33 % of dead grapevine materials using microscopy, plating assays and PCR with Botryosphaeriaceae multi-species primers (Bot100F/Bot472R). Isolations also showed that 15 to 68 % of the canes were internally infected by Botryosphaeriaceae species, with Neofusicoccum luteum and N. parvum being the most predominant species. Incidence varied between nurseries, with Nursery 5 having highest N. luteum incidence (53 %) and Nursery 3 having highest N. parvum incidence (88 %). Botryosphaeriaceae DNA was detected in rain-water run-off from the mother-vine canopy, but not in soil samples collected around infected mother-vines. However, samples from the nursery propagation stages had no visible or viable Botryosphaeriaceae propagules although PCR using multi-species primers detected Botryosphaeriaceae DNA from wash and hydration tanks, on grafting tools and in callusing media from the different nurseries. The single-stranded conformational polymorphism (SSCP) was able to identify N. parvum, N. luteum and Diplodia mutila as the most common species present in the nursery system. Overall results indicated that the canes grown in mother-vine blocks were the most likely source of inoculum for infection of young grafted plants by Botryosphaeriaceae species.