Wild and ancient wheat are considered to be a rich source of nutrients and better stress tolerant, hence being re-considered for mainstreaming its cultivation by the farmers and bringing it back to the food basket. In the present study, thirty-four diverse accessions of Indian dwarf wheat, Triticum sphaerococcum conserved in the Indian National Genebank were evaluated for thirteen-grain quality parameters namely thousand-grain weight (TGW), hectolitre weight (HW), sedimentation value (Sed), grain hardness index (HI), protein (Pro), albumin (Alb), globulin (Glo), gliadin (Gli), glutenin (Glu), gluten, lysine (Lys), Fe2+ and Zn2+ content, and four antioxidant enzymes activities. Substantial variations were recorded for studied traits. TGW, HW, Sed, HI, Pro, Alb, Glo, Gli, Glu, Gluten, Lys, Fe2+, and Zn2+ varied from 26.50-45.55 g, 70.50-86.00 kg/hl, 24.00-38.00 ml, 40.49-104.90, 15.34-19.35%, 17.60-40.31 mg/g, 10.75-16.56 mg/g, 26.35-44.94 mg/g, 24.47-39.56 mg/g, 55.33-75.06 mg/g, 0.04-0.29%, 42.72-90.72 ppm, and 11.45-25.70 ppm, respectively. Among antioxidants, peroxidase (POX), catalase (CAT), glutathione reductase (GR), and superoxide dismutase (SOD) activity ranged from 0.06-0.60 unit/ml, 0.02-0.61 unit/ml, 0.11-2.26 unit/ml, and 0.14-0.97 unit/ml, respectively. Hardness Index was positively associated with Pro and Zn2+ content whereas Lys was negatively associated with gluten content. Likewise, gluten and Fe2+ content had a positive association with the major protein fraction i.e., Gli and Glu. Hierarchical cluster analysis grouped 34 accessions into four clusters and the major group had nine indigenous and eight exotic accessions. We also validated high GPC accessions and EC182958 (17.16%), EC187176 and EC182945 (16.16%), EC613057 (15.79%), IC634028 (15.72%) and IC533826 (15.01%) were confirmed with more than 15% GPC. Also, superior trait-specific accessions namely, EC187167, IC534021, EC613055, EC180066, and EC182959 for low gluten content and IC384530, EC313761, EC180063, IC397363, EC10494 for high iron content (>76.51) were identified that may be used in wheat quality improvement for nutritional security of mankind.