In the face of climate changes and limited water availability for irrigated crop production, enhanced drought tolerance and adaptation is vital to improve wheat productivity. The objective of this study was to determine the responses of newly bred and advanced mutant lines of wheat based on agronomic traits and biomass allocation under drought-stressed and non-stressed environments for production and breeding. Fifty-three mutant lines, including the parental check and six check varieties, were evaluated under non-stressed (NS) and drought stressed (DS) conditions in the field and controlled environments using a 20 x 3 alpha lattice design with two replicates. The following agronomic data were collected: days to 50% heading (DTH), days to maturity (DTM), plant height (PH), number of productive tillers (PTN), shoot biomass (SB), root biomass (RB), total biomass (TB), root: shoot ratio (RSR), spike length (SL), thousand seeds weight (TSW) and grain yield (GY). Data were analyzed and summarized using various statistical procedures and drought tolerance indices were computed based on grain yield under NS and DS conditions. Significant (P < 0.05) differences were recorded among the mutant lines for most assessed traits under NS and DS conditions. Grain yield positively and significantly (p < 0.001) correlated with PTN (r = 0.85), RB (r = 0.75), SB (r = 0.80), SL (r =0.73), TB (r = 0.65), and TSW (r = 0.67) under DS condition. Principal component analysis revealed three components contributing to 78.55% and 77.21% of the total variability for the assessed agronomic traits under DS and NS conditions, respectively. The following traits: GY, RB, SB, and PTN explained most of the variation with high loading scores under DS condition. Geometric mean productivity (GMP), mean productivity (MP), harmonic mean (HM), and stress tolerance index (STI) were identified as the best drought tolerance indices for the identification of tolerant lines with positive correlations with GY under NS and DS conditions. Among the advanced lines tested, LMA16, LMA37, LMA47, LMA2, and LMA42 were selected as the superior lines with high performance and drought tolerance. The selected lines are recommended for multi-environment trails and release for production in water-limited environments in South Africa.