Abstract: Downy mildew (Plasmopara viticola) is the main grapevine disease in humid regions. In the present investigation, marker-assisted selection (MAS) was used to develop grapevine lines homozygous in loci Rpv1 and Rpv3 for resistance against P. viticola. The experimental populations UFSC-2013-1 (n = 420) and UFSC-2013-2 (n = 237) were obtained by self-pollination of two F1 full-sib plants, originated from a cross between two distinct breeding lines containing the downy mildew resistance loci Rpv1 and Rpv3 in heterozygosity. The two experimental populations were genotyped with four microsatellite markers flanking the two downy mildew resistance loci. Among 637 genotyped plants, 300 (48.2%) were homozygous for at least one resistance locus and 10 (1.57%) were homozygous for both Rpv1 and Rpv3 loci. These 10 plants challenged with P. viticola inoculum showed a clearly enhanced level of resistance. These plants have a great potential as resistance donors in grapevine breeding.