Wheat (Triticum aestivum L.) is one of the most important food crops feeding 36% of the world population. Yellow rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the devastating diseases and is a major biotic constraint in efforts to sustain global wheat production. The most effective means of managing yellow rust is the development and deployment of resistant wheat varieties. Transferring novel sources of stripe rust resistance genes from the wild species to cultivated varieties is an effective strategy to mitigate the disease. An introgression line (TSD858) derived from the cross T. spelta acc. IARI276/Agra Local BC1F9 showed high level field resistance (0-Immune reaction) against yellow rust pathogen. Inheritance of adult plant resistance was carried out in F2 and F2:3 populations. F2 population (TSD858 X HD2932) segregated as 131 resistant and 41 susceptible plants showing goodness of fit to 3:1 ratio (chi square= 0.124, p value= 0.725). F2:3 population (Agra Local X TSD858) segregated as 59 homozygous resistant, 82 segregating and 49 homozygous susceptible families showing goodness of fit to 1:2:1 ratio (chi square= 4.611, p value= 0.099). The results revealed that yellow rust resistance in TSD858 is due to presence of a single dominant gene. In order to confirm the novelty of this yellow rust resistance gene molecular marker analysis was done. Since Yr5 is the only gene reported from T. spelta associated markers were used. Yr5 gene is mapped to 2BL chromosome and is reported to be linked to microsatellite marker Xwmc175 (McGrann et al., 2014) and STS (Sequence tagged site) marker combinations STS7/8 and STS9/10 which is designed on the basis of the sequences of Xwgp-17 and Xwgp-18 markers identified using the resistance gene analog polymorphism (RGAP) technique (Chen et al., 2003). Using the set of primers STS-7 and STS-8, non Yr5 genotypes produces a band of 472 bases and Yr5 possessing genotypes showed a band of 478 bases. The other marker STS-9/STS-10 yields a product of 433 for non Yr5 genotypes and 439 for Yr5 positive genotype. However, both primer combinations showed no polymorphism in the tested lines. So as to detect polymorphism STS markers were converted to CAPS marker by performing restriction digestion of amplicon with Dpn II restriction enzyme. After digestion the Yr5 containing genotypes yields five restriction fragments (289 bp, 63 bp, 56 bp, 20 bp, and 12 bp), while non-Yr5 genotypes produce four restriction fragments (182 bp, 102 bp, 20 bp, and 12 bp). TSD858 and its donor T. spelta accession exhibited the same pattern as negative control proving its distinctness from Yr5. Hence this yellow rust resistance gene will be useful in resistance breeding improving diversity.