Leymus mollis Trin. (2n = 4x = 28, NsNsXmXm), an important wild relative of common wheat (Triticum aestivum L.), harbors abundant genes for disease resistance and excellent agronomic traits genes, representing a valuable genetic resource for wheat improvement. Cytological observation and genomic in situ hybridization (GISH) analyses confirmed that both lines contain 40 wheat chromosomes and a pair of L. mollis chromosomes. Fluorescence in situ hybridization (FISH) revealed that DM160 lacked chromosome 6B and exhibits structural variations in chromosome 5A and 7B, while DM252 lacks chromosome 6D and carries a mutated chromosome 7B. Combined molecular markers and liquid array analyses confirmed DM160 was a wheat–L. mollis 6Ns (6B) disomic substitution line, and DM252 was a wheat–L. mollis 6Ns (6D) disomic substitution line. Agronomic and disease resistance evaluations demonstrated that DM160 produces more tillers and exhibits stripe rust resistance, whereas DM252 displays reduced plant height, and confers resistance to both powdery mildew and stripe rust. Transcriptome sequencing revealed distinct gene expression profiles among DM160, DM252, and 7182, leading to the identification of six key candidate disease resistance genes. Furthermore, 15 DNA markers specific to Lm#6Ns were developed to track this chromatin in wheat backgrounds. These novel substitution lines provide valuable germplasm for enhancing disease resistance and agronomic traits in wheat, and serve as essential materials for further characterization and utilization of Lm#6Ns chromosomes.