In summary, we report phenotypic, genetic and genomic information regarding the interaction between the pathogen D. ampelina and grapevines, including two novel resistance loci, Rda1 and Rda2 located on chromosome 15 of V. cinerea B9 and chromosome 7 of ‘Horizon’, respectively. In the case of Rda1, our results suggest that the D. ampelina—V. cinerea B9 interaction is mediated by the action of one or more NB-LRR genes with homology to VIT_15s0046g02730 and VIT_15s0046g02800, providing the first hints about the molecular basis of the Phomopsis resistance phenotype.