T1RS.1BL wheat-rye translocation has been widely used in wheat breeding owing to agronomically beneficial genes located on the short arm of rye chromosome 1 (1RS). Although numerous methods have been developed to identify wheat lines possessing 1RS, there are some limits to their practical application in large breeding populations. In this study, we developed 10 homoeologous markers specific to each short arm of chromosome 1 of common wheat and rye based on genome databases, and their specificity was validated using wheat aneuploid and rye chromosome addition lines. Furthermore, recombinant inbred lines (RILs) were developed by crossing T1RS.1BL and non-translocated wheat and screening with the developed markers. Seed storage protein analysis revealed that the RILs possessed high gluten quality, and recombination had occurred in gluten and gliadin gene coding loci, except for in the 1RS region. The root and shoot biomasses of the RILs under drought and recovery were measured, indicating superior drought tolerance of the T1RS.1BL lines. The markers and RILs developed in this study could be useful in breeding wheat-rye translocations