QTL-seq analysis identified three major QTLs conferring subgynoecy in cucumbers. Furthermore, sequence and expression analyses predicted candidate genes controlling subgynoecy. The cucumber (Cucumis sativus L.) is a typical monoecious having individual male and female flowers, and sex differentiation is an important developmental process that directly affects its fruit yield. Subgynoecy represents a sex form with a high degree of femaleness and would have alternative use as gynoecy under limited resource conditions. Recently, many studies have been reported that QTL-seq, which integrates the advantages of bulked segregant analysis and high-throughput whole-genome resequencing, can be a rapid and cost-effective way of mapping QTLs. Segregation analysis in the F2 and BC1 populations derived from a cross between subgynoecious LOSUAS and monoecious BMB suggested the quantitative nature of subgynoecy in cucumbers. Both genome-wide SNP profiling of subgynoecious and monoecious bulks constructed from F2 and BC1 plants consistently identified three significant genomic regions, one on chromosome 3 (sg3.1) and another two on short and long arms of chromosome 1 (sg1.1 and sg1.2). Classical QTL analysis using the F2 confirmed sg3.1 (R2 = 42%), sg1.1 (R2 = 29%) and sg1.2 (R2 = 18%) as major QTLs. These results revealed the unique genetic inheritance of subgynoecious line LOSUAS through two distinct major QTLs, sg3.1 and sg1.1, which mainly increase degree of femaleness, while another QTL, sg1.2, contributes to decrease it. This study demonstrated that QTL-seq allows rapid and powerful detection of QTLs using preliminary generation mapping populations such as F2 or BC1 population and further that the identified QTLs could be useful for molecular breeding of cucumber lines with high yield potential.