Stripe rust, which is caused byPuccinia striiformisf. sp.tritici, is one of the most devastating foliar diseases of common wheat worldwide. Breeding new wheat varieties with durable resistance is the most effective way of controlling the disease. TetraploidThinopyrum elongatum(2n = 4x = 28, EEEE) carries a variety of genes conferring resistance to multiple diseases, including stripe rust, Fusarium head blight, and powdery mildew, which makes it a valuable tertiary genetic resource for enhancing wheat cultivar improvement. Here, a novel wheat–tetraploidTh. elongatum6E (6D) disomic substitution line (K17-1065-4) was characterized using genomic in situ hybridization and fluorescence in situ hybridization chromosome painting analyses. The evaluation of disease responses revealed that K17-1065-4 is highly resistant to stripe rust at the adult stage. By analyzing the whole-genome sequence of diploidTh. elongatum, we detected 3382 specific SSR sequences on chromosome 6E. Sixty SSR markers were developed, and thirty-three of them can accurately trace chromosome 6E of tetraploidTh. elongatum,which were linked to the disease resistance gene(s) in the wheat genetic background. The molecular marker analysis indicated that 10 markers may be used to distinguishTh. elongatumfrom other wheat-related species. Thus, K17-1065-4 carrying the stripe rust resistance gene(s) is a novel germplasm useful for breeding disease-resistant wheat cultivars. The molecular markers developed in this study may facilitate the mapping of the stripe rust resistance gene on chromosome 6E of tetraploidTh. elongatum.