Fourteen cucumber lines were tested for genetic homozygosity and performed pairwise comparison to identify a pair with thehighest DNA polymorphic level. Cucumber accessions CSL0067 and CSL0139 were selected to generate 315 F₂ populations. The geneticlinkage map based on 66 polymorphic SSR markers was constructed. It composed of eight linkage groups (LGs) spanning 474.4 cM.Downy mildew disease reaction was evaluated in cotyledons, first and second true leaf on 7, 10, and 14 day after inoculation. The resultsshowed that downy mildew resistance was controlled by multiple recessive genes. The susceptible to resistant ratio of F₂ progenies fit 9:7 susceptible/resistant segregation types corresponding to duplicate recessive epistasis. Fourteen QTLs were detected. The phenotypicvariance ranged from 5.0 to 12.5%, while LOD values ranged from 3.538 to 9.165. Two major QTLs and two QTL hotspots were identified.Moreover, the additive effects data explained that these QTL reduced downy mildew susceptibility.