Based on the similarity in gene structure between rice and wheat, the polymerase chain reaction (PCR)-based landmark unique gene (PLUG) system enabled us to design primer sets that amplify wheat genic sequences including introns. From the previously reported wheat PLUG markers, we chose 144 markers that are distributed on different chromosomes and in known chromosomal regions (bins) to obtain rye-specific PCR-based markers. We conducted PCR with the 144 primer sets and the template of the Imperial rye genomic DNA and found that 131 (91.0%) primer sets successfully amplified PCR products. Of the 131 PLUG markers, 110 (76.4%) markers showed rye-specific PCR amplification with or without restriction enzyme digestion. We assigned 79 of the 110 markers to seven rye chromosomes (1R to 7R) using seven wheat-rye (cv. Imperial) chromosome addition and substitution lines: 12 to 1R, 8 to 2R, 11 to 3R, 8 to 4R, 16 to 5R, 12 to 6R, and 12 to 7R. Furthermore, we located their positions on the short or long (L) chromosome arm, using 13 Imperial rye telosomic lines of common wheat (except for 3RL). Referring to the chromosome bin locations of the 79 PLUG markers in wheat, we deduced the syntenic relationships between rye and wheat chromosomes. We also discussed chromosomal rearrangements in the rye genome with reference to the cytologically visible chromosomal gaps.