Sustainable wheat production in drought prone areas can be achieved by developing resilient wheat varieties. In the present study, chemical mutagenesis was used to induce mutations in a cultivated wheat variety ‘NN-Gandum-1’. In total, 44 mutants were selected based on their high yield potential for exposing to well-watered (W1) and rainfed (W2) conditions for one season. Then 24 mutants were selected, and were exposed to W1 and W2 regimes. On the basis of least relative reduction in physiological parameters under W2 regime, five mutants were selected for conducting exome capturing assays. In total, 184 SNPs were identified in nine genes (ABC transporter type 1, Aspartic peptidase, Cytochrome P450, transmembrane domain, Heavy metal-associated domain, HMA, NAC domain, NAD (P)-binding domain, S-type anion channel, Ubiquitin-conjugating enzyme E2 and UDP-glucuronosyl/UDP-glucosyltransferase). Maximum number of mutations were observed in chr.2D, which contained mutations in three genes i.e. ABC transporter type 1, NAD (P)-binding domain and UDP-glucuronosyl/UDP-glucosyltransferase which may have a role in conferring drought tolerance. The selected mutants were further tested for studying their biochemical responses under both the regimes for two years. The extent of membrane damage was estimated through malondialdehydeand hydrogen per oxidase and tolerance to drought stress was assessed via antioxidant enzymes in leaves. The selected mutants under drought stress increased the accumulation of proline content, total soluble sugars, total free amino acids, while decreased total chlorophyll content, carotenoids and total soluble protein. Finally, the procedure of narrowing down the number of developed mutants from a large mutation population (>4000) is found useful for exploring the complex trait like drought without compromising yield potential. These mutants can further be explored to understand the genetic circuits of drought tolerance in wheat which will pave the way towards improving livelihood of resource poor farming community mostly relying on cereal food.