Stripe rust (caused by Puccinia striiformis) occurs annually in most wheat-growing areas of the world. Thinopyrum ponticum has provided novel rust resistance genes to protect wheat from this fungal disease. Wheat – Th. ponticum partial amphiploid line 7430 and a substitution line X005 developed from crosses between wheat and 7430 were resistant to stripe rust isolates from China. Genomic in situ hybridization (GISH) analysis using Pseudoroegneria spicata genomic DNA as a probe demonstrated that the partial amphiploid line 7430 contained ten Js and six J genome chromosomes, and line X005 had a pair of Js-chromosomes. Giemsa-C banding further revealed that both lines 7430 and X005 were absent of wheat chromosomes 6B. The EST based PCR confirmed that the introduced Js chromosomes belonging to linkage group 6, indicating that line X005 was a 6Js/6B substitution line. Both resistance observation and sequence characterized amplified region (SCAR) markers displayed that the introduced chromosomes 6Js were responsible for the stripe rust resistances. Therefore, lines 7430 and X005 can be used as a donor in wheat breeding for stripe rust resistance.