Puccinia graminis f. sp. tritici (Pgt) and P. triticina (Pt), the causal agents of stem rust and leaf rust, respectively form new physiological races that significantly reduce growth and yield of wheat cultivars. Therefore, seeking for resistant cultivars and exploring it to continuously produce new wheat cultivars resistant to stem and leaf rust through breeding programs is urgent. The aim of the present study was to assess 18 Egyptian wheat genotypes for resistance to stem and leaf rust. The 18 genotypes were also analyzed for polymorphism using 20 SCoT and SRAP primers. Furthermore, the activity of chitosan-cupper composite nanoparticle in controlling stem rust is examined and its mode of action is studied using molecular docking analysis. In seedling stage, the genotypes were tested against 20 stem rust races, and the host reaction types were noticed. The lowest host reaction types (It = 0; to 2++) were recorded for Sakha 94, Sakha 95, Beni Sweif 4, Beni Sweif 7, Sohag 4, Sohag 5 and Gemmeiza 12. These genotypes except Gemmeiza 12 were resistant to all races. The remaining genotypes were susceptible to most races, but Giza 160 was highly susceptible to all races. In adult stage, the 18 genotypes were evaluated for resistant to stem rust and leaf rust in two different location, i.e. Giza and sids. The evaluation was expressed as percentage of final rust severity (FRS%), Area under disease progress curve (AUDPC) and rate of rust disease increase (r-value). SCoT and SRAP analysis generated 140 and 121 polymorphic band with 97 and 99% polymorphism, respectively. Among them, 71 and 73 were unique loci for SCoT and SRAP, respectively. The 18 genotypes were divided into two main groups depending on the similarity matrix. The first cluster consists of the most resistant genotypes to leaf rust (Giza 171, Sakha 94, Misr 1, Misr 2, Misr 3, Giza 168, Gemmeiza 12 and Sids 12) in addition to two cultivars susceptible to leaf and stem rust (Beni Sweif 7, Gemmeiza 11). Meanwhile, the second ones consists of the most susceptible genotypes to stem and leaf rust (Giza 164, Sakha 69, Giza 160, Beni Sweif 4, Sohag 5 and Sohag 4) in addition to Sakha 95 (resistant to leaf rust but susceptible to stem rust) and Shandaweel 1 (resistant to stem rust but susceptible to leaf rust). Moreover, the 18 genotypes were sprayed with Cu-chitosan composite nanoparticle either before or before and after inoculation with uridiospores of stem rust to determine the effect of this solution and its application method in controlling the disease. The infection was reduced when the plant sprayed 24 h before and 24 h before and after inoculation. Incubation and latent periods were increased in treated plant genotypes. Besides, the treatment gave the lowest infection type compared to the control. The foliar spray application didn’t affect the efficacy of the tested treatment.