Cucumber is a typical monoecious plant with individual male and female flowers, and sex expression in cucumber is mainly determined by three major genes: F/f, M/m and A/a. Gynoecy plays an important role in cucumber hybrid breeding and use of gynoecious lines as maternal parent ensures high productivity. The purpose of this study is to identify a co-dominant molecular marker linked to F locus to distinguish homozygous and heterozygous gynoecious plants for cucumber breeding programme. Firstly, we analyzed the sequence polymorphism of 5 gynoecious and 5 monoecious inbred lines to detect polymorphism to develop the marker linked to F locus. A pair of specific primer based on insertion/deletion polymorphism on branched-chain amino acid transaminase (BCAT) gene was designed and examined the polymorphism in the parents, F1 and F2 segregating population derived from gynoecious (WJEF11) and monoecious (WNEF8) inbred lines. The result showed that the specific fragment amplified with Cs-Female-F/Cs-Female-R, was identified as a co-dominant marker and co-segregated with sex phenotype in F2 population. Furthermore, we present a new linkage map for F locus using Indel markers. This is the first report for the development of F locus specific co-dominant marker which can distinguish homozygous and heterozygous gynoecious and it could be used in marker-assisted selection in cucumber breeding.