Rye (Secale cereale L., 2n = 2x = 14, RR) is regarded as a significant genetic resource for improving common wheat because of its resistance to multiple diseases and abiotic-stress tolerant traits. The 1RS chromosome from the German cultivated rye variety Petkus is very important in wheat breeding, but its weakened disease resistance demands the identification of new resources. In the present study, a novel derived line called D27 was developed from common wheat and Mexico Rye. Cytological observations characterized the karyotype of D27 as 2n = 42 = 21II. Genomic in situ hybridization indicated that a pair of whole-arm translocated chromosomes were inherited normally in the mitotic and meiosis stages of D27. Experiments using fluorescence in situ hybridization (FISH) and gliadin electrophoresis showed that D27 lacked its wheat 1DS chromosomes and they were replaced by 1RS chromosomes of Mexico Rye, which was supported by wheat simple-sequence repeat markers, rye sequence characterized amplified region markers and wheat 40K SNP array analysis. The wheat 1DS chromosomes could not be detected by molecular markers and wheat SNP array, but the presence of rye 1RS chromosomes was confirmed. Agronomic trait assessments indicated D27 had higher tiller number, enhanced stripe rust and powdery mildew resistance. Dough properties analysis showed that the replacement of 1DS leaded to higher viscosity and lower elasticity of dough in D27, which was benefit for cake making. In conclusion, the novel cytogenetically stable common wheat-Mexico rye T1DL·1RS translocation line D27 can be used as an outstanding germplasm in wheat disease resistance and yield improvement breeding programs, and in genetic diversity research of rye 1RS chromosome.