The downy mildew disease caused by the oomycetePlasmopara viticolais a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locusRpv12, mediating resistance againstP. viticola, was originally found in AsianVitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploidRpv12-carrier Gf.99-03 was created and annotated. The defense response againstP. viticolawas investigated in an infection time-course RNA-seq experiment, revealing approximately 600 upregulatedVitisgenes during host–pathogen interaction. TheRpv12regions of the resistance and the sensitivity encoding Gf.99-03 haplotype were structurally and functionally compared with each other. Two different clusters of resistance-related genes were identified within theRpv12locus. One cluster carries a set of four differentially expressed genes with threeACCELERATED CELL DEATH6-like genes. The other cluster carries a set of six resistance gene analogs related to qualitative pathogen resistance. TheRpv12locus and its candidate genes forP. viticolaresistance provide a precious genetic resource forP. viticolaresistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to theR-genes enable its improved applicability in marker-assisted grapevine breeding.