Transforming resistance genes of wild relatives into elite cultivars is an effective approach to improve the wheat disease resistance. Identification of the alien chromosomes by deployment of a rapid and precise method becomes important in order to simplify the process on selection and utilization of the favorable genes. In this study, CH357, a new wheat germplasm resource which was derived from wheat-Thinopyrum intermedium partial amphiploid TAI7047, was subjected for tests upon infections of Powdery mildew and Stripe rust, as well as the evaluation by fluorescent in situ hybridization (FISH) and molecular markers detection. The results showed that CH357 contained a wheat-Th. intermedium 6JS/6B chromosome substitution. This genotype was tested to be resistant against wheat powdery mildew and stripe rust, and the resistances were likely caused by 6JS chromosome of Th. Intermedium, thus raising a potential for wheat resistance breeding. Furthermore, 160 STS markers were developed based on the Contigs sequences of Th. intermedium group 6 genome. Out of that, eight markers were found to specifically amplify the alien chromosome of CH357. The PCR-based assay will provide a user-friendly method for identifying the 6JS chromosome or fragments of Th. intermedium in wheat.