Leaf senescence is a notably important trait that limits the yield and biomass accumulation of agronomic crops. Therefore, determining the chromosomal position of the expression sequence tags (ESTs) that are associated with leaf senescence is notably interesting in the manipulation of leaf senescence for crop improvement. A total of 32 ESTs that were previously identified during the delaying leaf senescence stage in the stay-green wheat cultivar CN17 were mapped to 42 chromosomes, a chloroplast, a mitochondrion, and a ribosome using in silico mapping. Then, we developed 19 pairs of primers based on these sequences and used them to determine the polymorphisms between the stay-green cultivars (CN12, CN17, and CN18) and the control cultivar MY11. Among the 19 pairs of primers, 5 pairs produced polymorphisms between the stay-green cultivar and the non-stay-green control. Further studies of Chinese Spring nullisomic-tetrasomics show that JK738991 is mapped to 3B, JK738983 is mapped to 5D, and JK738989 is mapped to 2A, 4A, and 3D. The other two ESTs, JK738994 and JK739003, were not assigned to a chromosome using the Chinese Spring nullisomic-tetrasomics, which indicates that these ESTs may be derived from rye DNA in the wide cross. In particular, the ESTs that produce polymorphisms are notably useful in identifying the stay-green cultivar using molecular marker-assisted selection. The results also suggest that the in silico mapping data, even from a comparison genomic analysis based on the homogeneous comparison, are useful at some points, but the data were not always reliable, which requires further investigation using experimental methods.