Drought is a grave concern due to changing climate phenomenon which drastically affects wheat production throughout the world. Screening drought tolerant varieties of wheat are important for ameliorating productivity of water scarce areas. An experiment was conducted at physiology division Nuclear Institute of Agriculture Tandojam (Latitude: 25.433 Longitude: 68.533) Sindh, Pakistan during 2016-2017. Treatment included a factorial combination using a completely randomized design (CRD) with three replications. Six wheat cultivars (cv. cultivars IBWSN-1010, IBWSN-1025, TD-1, ESW-9525, Khirman and Chakwal-86) were investigated for their response at seedling stage under different water stress treatments (T-1 0, T-2 -0.5, T-3 -0.75 and T-4 -1.0 MPa) in controlled conditions. The results of experiments with reference to cultivars revealed that cultivar Khirman & IBWSN-1010 showed maximum shoot length (17.33 and 16.68 cm), while the cultivar Khirman and TD-1showed maximum root length (10.02 and 8.67 cm), shoot fresh weight (34.46 g 10^-1shoots), root fresh wt. (71.76 g 10^-1shoots), shoot dry wt. (13.55 g 10^-1shoots), root dry wt. (13.62 g 10^-1roots),while the cultivar IBWSN-1010 observed more chlorophyll contents (0.27 mg g1 fresh wt) and ionic contents (K⁺ and Ca2⁺, and K⁺ /Ca2⁺ratio)(1.05, 1.03 and 0.93 %). Among all the wheat cultivars tested Khirman and IBWSN-1010 are tolerant cultivar sand have the potential to perform better under drought conditions, whereas IBWSN-1025 and Chakwal-86 were moderately tolerant under water stress conditions. Moreover, the cultivars i.e. TD-1 and ESW-9525 are the sensitive cultivars under drought environment. It may be concluded from present in-vitro studies that osmotic stress significantly reduced the shoot/root length fresh and dry weight in all six wheat cultivars. The maximum reduction (P≤0.05) was found at higher osmotic stress induced by PEG-6000 (-1.0 MPa).