The wild relative’s diploid species, which are reproductively isolated from one another, compromise populations with marked morphological variation, wide climatic tolerance, and adaptation to diverse habitats, and also vary genetically in biotic, abiotic stresses, and in seed protein content and quality. Large-scale proteomic analysis of three wild relatives of wheat grain (AA, BB, and DD genome) using matrix assisted laser desorption/ionization- time of flight (MALDI-TOF-MS), multi-dimensional protein identification technology (MudPIT), allowed the detection and classification of 213, 255 unique proteins (peptide match ≥ 2), which represents the most wide-ranging proteome exploitation to date. Development of standard proteomes exhibiting all of the proteins involved in normal physiology will facilitate the delineation of disease/defense (no. of unique protein (n) =33, 51), metabolism (n=15, 32), energy metabolism (n= 21, 27), protein synthesis (n=16, 22), folding/stability (n=17, 18), transcription (n=6, 18), cell growth/division (n=17, 17), signal transduction (n=16, 15), cellular organization (n=11, 12), development (n=9, 9), storage protein (n= 30, 7), transport facilitation (n=8, 6), and unclear classification (n= 14, 21), which is identification by using MALDI-TOF and LCQ DECA mass spectrometry couple to mascot database search, respectively. For instance, ABA inducible protein PHVA1 (HVA1), which can be induced by drought, cold, heat and salinity condition, and also basic endochitinase (RSCC, RSCA) showed defense against chitin containing fungal pathogens. Gluten (glutenin and gliadin), which is very important determinant for making high quality bread, noodles, and also associated with visco-elasticity. By using MALDI-TOF, we identified abundant disease related protein such as NBS-LRR involves in response to the presence of a foreign body or the occurrence of an injury, which result in restriction of damage to the organism attacked or prevention/recovery from the infection caused by the attack, puroindoline (a & b) and grain softness protein represents the molecular-genetic basis of grain texture. In addition, the PIN A and PIN B gene products have anti microbial properties with potential role in plant defense. Recent advances in mass spectrometry and bioinformatics have provided the means to characterize complex protein landscapes from a wide variety of organisms. Hierarchical clustering could be applied to protein information from different samples using Gene Pattern and NCSS software. Here we report also genome specific protein interaction network using Cytoscape software, which provides further insight into the molecular mechanism of biochemical pathways. By integrating shotgun proteomics with statistical and computation alanalyses, we developed promising understand about expressed protein and protein functions. Our approach should be applicable for marker assisted breeding or genetransfer for quality and stress research of cultivated wheat.