The invention discloses gene segments linked with yellow and green cotyledons of cucumbers and application. The gene segment linked with yellow cotyledons of cucumbers is a nucleotide sequence shown in SEQ ID No.1, and the gene segment linked with green cotyledons of cucumbers is a nucleotide sequence shown in SEQ ID No.2. The gene segment linked with yellow cotyledons of cucumbers and the gene segment linked with green cotyledons of cucumbers are used for detecting the constructed F2-generation segregation population and cucumber germplasm resources, the identification shows that the coincidence rate of the detection result and the artificial phenotype reaches 96.77%, and the linkage inheritance distance of the genes related to the colors of cucumber cotyledons is 3.2cm. The invention has the advantages of rapidness, accuracy, no influence of environmental conditions and the like, and has a great application value for molecular marker-assisted breeding of cucumbers.