Thinopyrum intermedium (2n = 6x = 42, JJJSJSStSt) has been hybridized extensively with common wheat and proven to be a valuable germplasm source for improving disease resistance and yield potential of wheat. A novel disease-resistant wheat-Th. intermedium double substitution line X479, carrying 1St(1B) and 4St-4JS (4B), was identified using multi-color non-denaturing fluorescence in situ hybridization (ND-FISH). With the aim of transferring Thinopyrum-specific chromatin to wheat, a total of 573 plants from F2 and F3 progenies of X479 crossed with wheat cultivar MY11 were developed and characterized using sequential ND-FISH with multiple probes. Fifteen types of wheat-Thinopyrum translocation chromosomes were preferentially transmitted in the progenies, and the homozygous wheat-1St, and wheat-4JSL translocation lines were identified using ND-FISH, Oligo-FISH painting and CENH3 immunostaining. The wheat-4JSL translocation lines exhibited high levels of resistance to stripe rust prevalent races in field screening. The gene for stripe rust resistance was found to be physically located on FL0–0.60 of the 4JSL, using deletion lines and specific DNA markers. The new wheat-Th. intermedium translocation lines can be exploited as useful germplasms for wheat improvement.