Wheat is grown in rainfed areas characterized by erratic rainfall with a high probability of drought spells during the vegetative and reproductive growth. Drought poorly affects crop growth and yield by altering metabolic and physiological processes. Plants tolerate drought through osmotic adjustment, stomatal resistance, increased water uptake and accumulation of waxy layer. Cultivars possessing drought tolerant traits may be promising candidates for drought prone regions. The present study was planned for molecular, physiological and biochemical evaluation of wheat cultivars for drought tolerance. Four experiments were accompanied for this purpose. In first experiment wheat seeds of five cultivars (GA-2002, Chakwal-97, Uqab- 2000, Chakwal-50 and Wafaq-2001) were exposed to osmotic stress (-2, -4, -6, and -8 bars) created by soaking the blotters with different concentrations of PEG-6000. Assessment of cultivars was based on germination percentage, speed of germination, coleoptile length, root-shoot length, dry and fresh weight. In second laboratory experimentation, sterilized seeds were sown in petri plates and after seven days the seedlings were transplanted into hydroponics having modified MS medium solution followed by its supplementation by PEG-6000 to induce osmotic stress of -2, -4, -6 and -8 bars at 3-4 days interval. After two days, plants exhibited visible effects of stress. Thus the plants were harvested for analysis and data collection. Evaluation of cultivars was constructed on proline, sugar, chlorophyll, protein, amino acids, root- shoot dry and fresh weight basis. All cultivars exhibited decline in germination percentage, speed of germination, root-shoot length, coleoptile length, seedling fresh and dry weight under water stress environments. Total soluble sugar, soluble protein, amino acids and proline have direct relationship with osmotic stress while total chlorophyll contents, seedling fresh and dry weight have inverse association with water stress. Third experiment was carried out by employing different levels of field capacity (80%, 60%, 40% and 20%) as stress sources in glass house/plant shed. Cultivars were assessed on the basis of Gas exchange (photosynthesis rate, transpiration rate and stomatal conductance) measurements, epicuticular wax, relative water content and osmotic potential. With increase in water stress photosynthesis rate, transpiration rate, stomatal conductance, relative water content and osmotic potential drops however epicuticular wax content increased with an increase in water stress conditions. At the end for drought response, wheat cultivars were evaluated on molecular basis. A set of specific primers (TdDHN15, WDHN13, TdDHN16, TdDHN9.6) were used for presence and expression of dehydrin genes which can confer drought resistance to a genotype. RNA was extracted by Trizol method. Then first strand cDNA synthesis was done by using superscript II RT. PCR was carried out by using the cDNA. PCR products were separated on Agrose gel and visualized under gel doc system. Chakwal-50 followed by GA-2002 and Chakwal-97 performed best at various stress levels for germination percentage, speed of germination, root-shoot fresh and dry weight, total chlorophyll contents, proline, soluble protein, total soluble sugars contents, photosynthesis rate, transpiration rate, stomatal conductance, wax content, osmotic potential and relative water contents. However performance of Wafaq-2001 and Uqab-2000 was poor. The gene expression of WDHN13 was observed only in GA- 2002 and Chakwal-50. None of the other gene expression was recorded in any other cultivars. It can be concluded that water stress levels had substantial effects on germination and seedling growth. Morphological, biochemical, physiological and molecular analysis revealed that adequate genetic difference for drought tolerance existed in wheat genotypes tested. Chakwal-50 and GA-2002 may prove a promising parent material for breeding drought tolerant wheat cultivars. Further molecular investigations are suggested to assess the genetic basis of drought tolerance. Chakwal- 50 and GA-2002 may be considered better genotypes for low rainfall drought prone areas.