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The rye (<i>Secale cereale</i> L.) 5R chromosome contains some elite genes that can be used to improve wheat cultivars. In this study, a set of 5R^Ku dissection lines was obtained, and 111 new PCR-based and 5R^Ku-specific markers were developed using the specific length amplified fragment sequencing (SLAF-seq) method. The 111 markers were combined with the 52 5R^Ku-specific markers previously reported, and 65 <i>S. cereale</i> Lo7 scaffolds were physically mapped to six regions of the 5R^Ku chromosome using the 5R^Ku dissection lines. Additionally, the 5RL^Ku arm carried stripe rust resistance gene(s) and it was located to the region L2, the same region where 22 5R^Ku-specific markers and 11 <i>S. cereale</i> Lo7 scaffolds were mapped. The stripe rust resistance gene(s) located in the 5RL^Ku arm might be new one(s) because its source and location are different from the previously reported ones, and it enriches the resistance source of stripe rust for wheat breeding programs. The markers and the <i>S. cereale</i> Lo7 scaffolds that were mapped to the six regions of the 5R^Ku chromosome can facilitate the utilization of elite genes on the 5R chromosome in the improvement of wheat cultivars.