AbstractCommercial production of spinach (Spinacia oleraceaL.) is centered in California and Arizona in the US, where downy mildew caused byPeronospora effusais the most destructive disease. Nineteen typical races ofP. effusahave been reported to infect spinach, with 16 identified after 1990. The regular appearance of new pathogen races breaks the resistance gene introgressed in spinach. We attempted to map and delineate theRPF2locus at a finer resolution, identify linked single nucleotide polymorphism (SNP) markers, and report candidate downy mildew resistance (R) genes. Progeny populations segregating forRPF2locus derived from resistant differential cultivar Lazio were infected using race 5 ofP. effusaand were used to study for genetic transmission and mapping analysis in this study. Association analysis performed with low coverage whole genome resequencing-generated SNP markers mapped theRPF2locus between 0.47 to 1.46 Mb of chromosome 3 with peak SNP (Chr3_1, 221, 009) showing a LOD value of 61.6 in the GLM model in TASSEL, which was within 1.08 Kb from Spo12821, a gene that encodes CC-NBS-LRR plant disease resistance protein. In addition, a combined analysis of progeny panels of Lazio and Whale segregating forRPF2andRPF3loci delineated the resistance section in chromosome 3 between 1.18–1.23 and 1.75–1.76 Mb. This study provides valuable information on theRPF2resistance region in the spinach cultivar Lazio compared toRPF3loci in the cultivar Whale. TheRPF2andRPF3specific SNP markers, plus the resistant genes reported here, could add value to breeding efforts to develop downy mildew resistant cultivars in the future.