A total of 57 Ilyonectria liriodendri isolates were identified by a combination of species-specific PCR and DNA sequencing from a collection of 174 Ilyonectria-like isolates recovered from 101 diseased grapevine samples. These samples were representative of the national vineyard, comprising material contributed by 49 grape growers across seven grape growing areas. This species was predominant, representing 33% of the recovered isolates, and has been reported as a major pathogen of grapevines in other countries. The genetic diversity of the 57 New Zealand isolates was compared to that of isolates from Australia and South Africa using universally primed polymerase chain reaction (UP-PCR). A total of 66 informative loci distinguished 52 genotypes, of which five contained up to four clonal isolates. Four main clades were identified in a neighbour-joining (NJ) tree. The international isolates (Australia and South Africa) were placed in a clade that did not include New Zealand isolates. There was a high level of intra- and inter-vineyard genetic variation indicating the free movement of isolates between regions. A subset of nine isolates from different branches of the NJ tree produced two vegetative compatibility groups and hyphal fusion was observed between non-self pairings. Pathogenicity tests using isolates from different genetic groups inoculated onto either detached roots or 1-year-old potted vines showed variability in virulence; however, no correlations were detected.