Drought is a major threat to wheat production worldwide and development of droughttolerant wheat genotypes may improve the production of this cereal crop in drought affected regions. In the present study, 24 mutant lines were developed by treating a wheat variety 'NN-Gandum-1(NN-1) with ethyl methane sulfonate (EMS). The M5, M6, and M7 generations were evaluated for quantitative traits under well irrigated and rain-fed conditions. Furthermore, best performing mutants of M6 and M7 generations were examined for biochemical parameters. A Substantial correlation was found among the quantitative traits (plant height, spike length, tillers per plant, number of spikelets per spike and thousand grain weight) which confirmed their high heritability and relative relation. Principal component analysis (PCA) showed that the first two PCs with eigen values >1 had more than 50% of the total variability among the mutant lines under normal and water limited conditions. Cluster analysis depicted that considerable variations existed among all mutant lines. Among these mutant lines, some mutants showed significant variation consistently larger than corresponding values of wild type in multi-generation replication. Using drought tolerance indices (Stress tolerance index and geometric mean productivity), high yielding mutant lines under drought stress were selected. Across all these three-mutant generations, Mutant lines NN1-M-363, NN1-M-506, NN1-M-700, NN1-M-701, and NN1-M-1621 showed significant improvement as compared to wildtype in response to stress conditions. The selected five drought tolerant mutants showed increased accumulation of proline contents, total soluble sugars, total free amino acids, while decreased total chlorophyll content, carotenoids, and total soluble proteins.Results suggested that the morphological traits and yield attributes along with biochemical parameters could be utilized for the evaluation of wheat cultivars under drought stress.