The productivity of wheat (Triticum spp.), an important staple food grain crop is under the constraints of drought stress. Molecular characterization through RAPD and SSR markers specific for drought stress in 22 wheat genotypes displayed two major clusters viz. Triticum aestivum L. and T. durum L. which was further divided into irrigated and rainfed. Principle component analysis was consistent with dendrograms. All the markers displayed PIC value greater than 0.5 showing its usefulness in detecting differences within the genotypes. Artificial stress induced by polyethylene glycol for biochemical and physiological characterization showed significant differences among the genotypes and drought levels which correlated with the molecular characterization. The proline content was increased during stress with the induction of stress specific proteins as revealed by SDS-PAGE. The tolerance of wheat genotypes to drought stress was assessed based on molecular markers, physiological observations and biochemical analysis.