Abstract Leymus mollis (Trin.) Pilger (NsNsXmXm, 2n = 28), a wild relative of common wheat, possesses many potentially valuable traits that could be transferred to common wheat during breeding programs. In this study, the karyotypic constitution of a wheat – L.mollis 3D(3Ns#1) disomic substitution line isolated from the F5 progeny of octoploid Tritileymus M842-16 × Triticum durum cv. D4286, which was designated as 10DM57, was determined using genomic in situ hybridization (GISH), fluorescent in situ hybridization (FISH), SSR markers, and EST-STS markers. Screening of mitosis and meiosis showed that 10DM57 had a chromosome karyotype of 2n = 42 = 21II. GISH indicated that 10DM57 was a line with 40 chromosomes from wheat and two of the Ns chromosomes from L. mollis, which formed a ring bivalent in pollen mother cells at metaphase I. FISH analysis showed that the chromosome 3D may be replaced by 3Ns#1 in 10DM57. DNA markers, including SSR and EST-STS primers, showed that the pair of wheat chromosome 3D in 10DM57 was substituted by the pair of chromosome 3Ns#1 from L. mollis. Evaluation of the agronomic traits showed that, compared with its common wheat relative 7182, 10DM57 was resistant to leaf rust while the spike length and number of spikes per plant were improved significantly, which correlated with a higher wheat yield. The new germplasm, 10DM57, could be exploited as an intermediate material in wheat genetic and breeding programs.