Plant productivity is greatly influenced by environmental stresses. Under stressful conditions, plants develop a plethora of biochemical and molecular mechanisms to cope with new situations that includes expression of numerous genes by signal transduction associated with stress tolerance. Plant osmotic stress responses are associated with changes in gene expression due to environmental stresses like drought, high salinity and low temperature. In the present study, our focus was on DNA fingerprinting of wheat (Triticum aestivum) germplasms of different genotypes viz Local Genotype, Mapping population, Synthetic and NIBGE on the basis of polymorphism. Various osmolytes and antioxidant enzymes were also studied in the drought affected genotypes followed by their association with SSR primers. Drought was induced in wheat samples by using polyethylene glycol (PEG 8000). Biochemical estimation of proline was done by spectrophotometric method and glycine- betaine and trehalose were analyzed on HPLC. The antioxidant enzymes viz superoxide dismutase, catalase and ascorbate peroxidase were determined through spectrophotometer method. Root/shoot ratio was enhanced in the drought affected genotypes. The levels of antioxidant enzymes were enhanced significantly in all the genotypes except those of NIBGE. Glycine-betaine, proline and trehalose promoted a positive effect in wheat germplasm under drought stress. For DNA fingerprinting, SSR marker studies was performed using 45 Ksum series primer pairs in the selected wheat genotypes. The DNA fingerprinting results have shown that the different wheat genotypes had association linked with osmoprotectants and antioxidant enzymes during drought stress. Statistical results have shown higher degree of marker-trait association among wheat genotypes and drought affected parameters. This study will strengthen the useful breeding programs especially to amplify efforts for continued genetic improvement of wheat to meet the growing requirements of an ever increasing population.