Root is important for improving crop adaptation to water deficit stress as it is the first organ to respond water deficit stress. But research on the impact of water deficit stress on root traits have been mostly neglected. In our study, a set of 98 chromosomal segment substitution lines (CSSLs) developed from a disomic substitution line (DS5U5A), where chromosome 5A in wheat cultivar WL711 is replaced with chromosome 5U of Ae. triuncialis acc pau3549. DS5U5A line have been crossed twice with pavon ph1b (a mutant of ph1b gene) and the plants with ph1b gene in homozygous form (ph1b ph1b) and chromosome 5U and 5A in heterozygous form have been selected and selfed upto BC2F6. This process induce homologous pairing between chromosome 5U and 5A. The BC2F6 CSSLs expected to carry different sized introgression of chromosome 5U in background of chromosome 5A. These CSSLs were evaluated for two consecutive years for root system architecture studies by sowing in field with three replications in randomized block design under drought as well as irrigated conditions. Among these CSSLs named 5U-23,5U-49 and 5U-96  were  identified as best lines having good root architecture (long, deep and thin roots and high root biomass) and better drought tolerance index. These CSSLs were also evaluated for different agronomic traits. Eight CSSLs named 5U-17, 5U-20, 5U-23, 5U-24, 5U-49, 5U-74, 5U-93, 5U-96 and 5U-98 have higher yield than popularly cultivated wheat varieties PBW 677 and PBW 1 Chapati under restricted irrigated conditions. CSSLs 5U-96 (1.047) has highest stress tolerance index followed by 5U-74 (0.776), 5U-24 (0.596), 5U-20 (0.592) and 5U-17 (0.557). These CSSLs were also amongst top ten lines for thousand kernel weight (g). Best 5U-5A CSSLs can further be used in wheat breeding programs to tailor genetic make-up of high yielding wheat cultivars that enables them to perform well under water deficit stress conditions.