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Powdery mildew is one of the most important diseases of cucumber (Cucumis sativus L.). For many years, researchers have studied and attempted to isolate resistance genes for this disease for breeding powdery mildew-resistant cucumber. In this study, bulked segregant analysis and an initial quantitative trait loci (QTLs) analysis based on a local linkage map were performed using an F2 population. A major locus on the long arm of chromosome 5 was identified for powdery mildew resistance and named pm5.1. For fine mapping of the pm5.1 locus, BC3F1 and BC2F2 populations that segregated only at the target genomic region were developed by marker-assisted selection. The location of the pm5.1 locus was confirmed by QTL analysis using a subset of the BC3F1 population and was delimited in a 1.8-cM interval between flanking markers UW065011 and UW065190. Using the large BC3F1 (n = 480) and BC2F2 (n = 483) populations, pm5.1 was mapped to a ~170-kb region between markers UW065021 and UW065094. Based on the annotations for the genes in this region, a MLO-like gene was identified as the most likely candidate for the pm5.1. Sequence alignment analysis of the MLO-like genes from susceptible and resistant cucumber lines revealed two types of mutations in this candidate gene that may result in recessive resistance to powdery mildew in the resistant cucumber lines.