Exploiting and utilizing excellent gene(s) from wild species has become an essential strategy for wheat improvement. In the disomic addition line 24-6-3, the 4Ns chromosomes from Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) carried valuable tiller and strip rust resistance gene(s), which was selected from the progeny of common wheat cv. 7182 and P. huashanica via embryo culture. Cytology, genomic in situ hybridization (GISH), and EST-STS analyses were used to detect the 4Ns chromosome in wheat background and its homoeologous relationship. Cytological studies demonstrated that 24-6-3 contained 44 chromosomes and formed 22 bivalents during meiotic metaphase I. GISH using P. huashanica genomic DNA as a probe indicated that a pair of Ns-chromosomes with strong hybridization signals had been introduced into 24-6-3. Ten EST-STS markers, i.e., BE404973, BE442811, BE446061, BE446076, BE497324, BE591356, BF473854, BG274986, BQ161513 and CD373484, which were located on the homoeologous group 4 chromosomes of wheat, amplified bands unique to P. huashanica in 24-6-3. This indicated the presence of an introgressed P. huashanica Ns chromosome pair belonging to homoeologous group 4, which we designated the 4Ns disomic addition line. After it was inoculated using mixed races of stripe rust in the adult stages, 24-6-3 expressed high stripe rust resistance, which was possibly derived from its P. huashanica parent. Moreover, its increased number of tillers was probably controlled by gene(s) located in P. huashanica chromosome 4Ns. These high levels of disease resistance and excellent agronomic traits make the 24-6-3 line a promising germplasm for breeding in wheat.