A dominantly inherited major-effect QTL for powdery mildew resistance in cucumber was fine mapped. Two tandemly arrayed cysteine-rich receptor-like protein kinase genes were identified as the most possible candidates. Powdery mildew (PM) is one of the most severe fungal diseases of cucumber (Cucumis sativus L.) and other cucurbit crops, but the molecular genetic mechanisms of powdery mildew resistance in cucurbits are still poorly understood. In this study, through marker-assisted backcrossing with an elite cucumber inbred line, D8 (PM susceptible), we developed a single-segment substitution line, SSSL0.7, carrying 95 kb fragment from PM resistance donor, Jin5-508, that was defined by two microsatellite markers, SSR16472 and SSR16881. A segregating population with 3600 F2 plants was developed from the SSSL0.7 × D8 mating; segregation analysis confirmed a dominantly inherited major-effect QTL, Pm1.1 in cucumber chromosome 1 underlying PM resistance in SSSL0.7. New molecular markers were developed through exploring the next generation resequenced genomes of Jin5-508 and D8. Linkage analysis and QTL mapping in a subset of the F2 plants delimited the Pm1.1 locus into a 41.1 kb region, in which eight genes were predicted. Comparative gene expression analysis revealed that two concatenated genes, Csa1M064780 and Csa1M064790 encoding the same function of a cysteine-rich receptor-like protein kinase, were the most likely candidate genes. GFP fusion protein-aided subcellular localization indicated that both candidate genes were located in the plasma membrane, but Csa1M064780 was also found in the nucleus. This is the first report of dominantly inherited PM resistance in cucumber. Results of this study will provide new insights into understanding the phenotypic and genetic mechanisms of PM resistance in cucumber. This work should also facilitate marker-assisted selection in cucumber breeding for PM resistance.