Psathyrostachys huashanica (2n = 2x = 14, NsNs) and Leymus mollis (2n = 4x = 28, NsNsXmXm) are important wild relatives of common wheat. The Ns chromosomes from two species have been successfully introgressed into wheat through distant hybridization. To compare the genetic effects and evolutionary relationship of Ns chromosomes from different genera in a wheat background, wheat-P. huashanica derivative WH15 and wheat-L. mollis derivative WM14-2 were selected. Sequential FISH-GISH showed that both WH15 and WM14-2 contained 40 wheat chromosomes (with 2D deletion) and two Ns chromosomes with different FISH karyotypes. Molecular markers and SNP array analysis revealed that the two lines both introduced 2Ns chromosomes. However, the P. huashanica 2Ns and L. mollis 2Ns had distinct sequence compositions, and the different SNPs between the two species 2Ns chromosomes were primarily clustered on the short arm. WH15 and WM14-2 exhibited significant differences in spike-related morphologies but shared leaf rust resistance and susceptibility to powdery mildew and Fusarium head blight. Cytogenetic analysis confirmed stable meiotic inheritance of the introduced 2Ns chromosomes. We further developed universal diagnostic markers for 2Ns chromosomes based on SLAF-seq. Therefore, substantial divergence likely exists between the Ns genomes of P. huashanica and L. mollis, and P. huashanica is probably not the direct Ns genome donor for Leymus. Our research-developed derivatives provide unique resources for comparative studies of the structural and functional evolution of homoeologous Ns chromosomes across genera, while offering valuable alleles for wheat improvement.