An efficient and reliable transformation system is imperative for improvement of important crop species like barley andwheat. Wheat transformation is complex due to larger genome size and polyploidy while barley has a limitation of genotypicdependency. The objective of current study was to compare the relative transformation efficiency of wheat and barley usingspecific expression vector pBRACT 214-NDPK2 constructed through gateway cloning carrying Nucleoside DiphosphateKinase 2 (NDPK2) gene. The vector was used to compare the transformation response in both crops using immatureembryos through Agrobacterium mediated transformation. Both wheat and barley showed different responses towards callusinduction and regeneration. Immature embryos of 1.5 to 2 mm in diameter was found optimum for wheat callus inductionwhile 1 to 1.5 mm for barley. Both embryogenic and non-embryogenic calli were found in wheat with significantly greatertendency for embryogenecity in barley. The overall regeneration response was found different for all transformed wheat andbarley cultivars. Wheat cultivars showed good response initially that drastically slowed down in later stages with theexception of Fielder that reached to the green shoots with good roots. The barley transformed lines showed goodregeneration response as compared to wheat. PCR analysis of putative transformants using genomic DNA showed amaximum of 27% transformation efficiency in barely. No true transformation response was obtained in all cultivars of wheatused in this study. The protocol developed for wheat and barley transformation will greatly be helpful in crop improvementprogramme through genetic engineering especially in diploid relatives of cereals.