In this paper, we report our study into the genetics of Phomopsis resistance of canes and clusters in three hybrid grapevine families. First, we quantified the segregation of cane and cluster symptoms in families derived from interspecific hybrids ‘Horizon’ and Illinois 547-1, V. vinifera ‘Chardonnay’, and V. cinerea B9. We used high-density genetic maps to study the association between phenotype and molecular markers, which allowed further identification of two novel major resistance loci. Candidate genes were further dissected through gene expression analysis.