Ethylene regulates sex expression in cucumber plants (Cucumis sativus L.). ACC synthase is a key factor during the ethylene biosynthesis. Pairs of PCR primers were synthesized corresponding to the conserved sequences of ACC synthase gene family. The 1 188 bp DNA fragment of ACC synthase gene (CS-ACS2) was amplified from genomic DNA of 8 different sexual phenotypes of cucumber respectively (GenBank accession number is DQ115884～DQ115886 and DQ115875～DQ115879). 8 SNPs have been identified by sequences analysis between 3 monoecious lines and 5 subgynoecious lines and gynoecious lines, which including 4 A←→G and 4 T←→C transition. Of these 8 SNPs, one locus is in intron and 7 loci in exons. Of the 7 SNPs located in exons, 3 SNPs are non-coding SNPs and 4 SNPs are coding SNPs (cSNPs) of which 3 induced changes of encoding amino acid of ACC synthase. The results of SNPs from subgynoecious lines and gynoecious lines suggest that single nucleotide mutation events of CS-ACS2 might be correlated with the development of subgynoecious lines and gynoecious lines in cucumber. Furthermore, CAPS marker C-MT705 was developed for identifying elite subgynoecious cultivar MT-705, which could be valuable in cucumber breeding. Besides, the SNPs and CAPS markers obtained in the study enriched molecular markers of cucumber.