Water stress is a primary constraint to achieve the goal of sustainable crop production. Water stress severely affects the seed production and oil yield of mustard genotypes. To overcome this problem, the development of water stress resilient mustard cultivars with potential seed and oil yield is a sustainable solution. Therefore, in vitro screening of mustard genotypes through PEG-6000 treatments were carried out on ten mustard genotypes. Along with control, two PEG-6000 levels were set to impose osmotic stress, such as, 6% and 10% PEG-6000. Data analyses depicted significant differences among all genotypes and between PEG-6000 treatments for all the studied traits, such as, shoot and root length, shoot and root fresh weight, shoot and root dry weight, K+ content, Ca++ content and K+/Ca++ ratio, demonstrating the availability of genetic differences in mustard genotypes for future stress breeding. Under PEG-6000 (6% and 10%) treatments, the genotypes like AARI-Canola, Khanpur Raya, Dhoom-1, Super Raya, Galaxy and Coral-432 exhibited high performance for seedling traits and less reduction due to PEG-6000 treatments. Hence, these mustard genotypes tend to provide useful genetic potential for water stress breeding. Keywords: Oilseed crops, genetic variations, drought stress, seed yield.