Downy mildew, caused by Pseudoperonospora cubensis (Berk. & Curt.) Rostov, is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber line CS-PMR1, derived from self-pollination of USDA Plant Introduction 197088, has a high level of resistance to downy mildew and is considered to be promising breeding material. In this study, we performed quantitative trait locus (QTL) analysis for downy mildew resistance using 111 recombinant inbred lines (RILs) derived from a cross between CS-PMR1 and the old Japanese cultivar Santou, which exhibits moderate resistance. The resistance of the RILs and their parents was evaluated by diverse methods using different plant organs (cotyledons, true leaves), stages (seedlings and adult plants), and evaluation criteria (lesion expansion and extent of sporulation). The high resistance of CS-PMR1 was associated with many QTLs with relatively small effects, whereas the moderate resistance of Santou was associated with one major QTL and possibly two others with relatively small effects. In all assays, the major QTL at which the Santou allele was associated with increased resistance had the largest effect. This QTL allele from Santou and several of the most effective QTL alleles identified in CS-PMR1 should be highest priority for selection to efficiently breed new cultivars that carry adequate levels of downy mildew resistance.