The invention discloses 17 EST sequence-based specific molecular markers for the chromosomes from 1R to 7R of rye, which are forward and reverse primers which are designed according to sequences of ESTs on the chromosomes of wheat and are specific markers for identifying the chromosomes from 1R to 7R of rye. The identification of the chromosomes from 1R to 7R of rye by using the molecular markers is implemented by the following basic steps: A, selecting and preparing marker primers; B, diluting the marker primers; and C, specific marking the chromosomes of the rye, namely extracting a template DNA, performing PCR amplification, detecting product of amplification and comparing results. In the invention, a set of specific molecular markers for the chromosomes of rye are designed on the basis of the sequences of the ESTs on the chromosomes of wheat, all chromosomes from 1R to 7R of rye in the genetic background of wheat can be identified quickly and accurately by using a common polymerase chain reaction (PCR) technique when the molecular markers are used, and a simple, convenient and effective molecular identification method is provided for transferring beneficial genes in the chromosomes of rye into a cultivation background of wheat.