The present study intended to assess the physiochemical and genetic responses of ten different wheat (Triticum aestivum L.) genotypes including bread wheat and synthetic hexaploids (SHs) under drought and control conditions. The experiment was performed in two factorial arrangements with genotypes as one and treatments as other factor. The drought treatment was applied at pre-anthesis stage by withholding water for 10-15 days and continued till the appearance of the symptoms of drought. The data for biochemical contents, physiological traits and gene expression was conducted in randomly selected wheat samples at the onset of drought symptoms. The enzymatic activity, proline and glycine betaine showed significant increase in wheat genotypes due to drought treatment, with maximum increase in SHs lines followed by bread wheat genotypes Galaxy-2013, Aas-2011 and AARI-2011. Conversely, all SHs recorded less reduction in physiological traits as compared to bread wheat genotypes due to application ofdrought stress. Besides correlation, PCA and heatmap analysis proved significantly high impact of drought on the correlation and expression of physiological and biochemical traits as compared to control treatment. Furthermore, in all wheat genotypes, the expression of drought associated genes TaDREB1A, TaGROS-A, TaERF3, TaCPK34, TaLEA3, TaHSFA1a, TaWRKY44, TaZFP34 and TaEXPA2changed complementary to the corresponding physiological and biochemical traits. The most significant change in expression of these genes with respect to corresponding physiological and biochemical traits under drought stress was recorded in SHs, Galaxy-2013, Aas-2011 and AARI-2011.The drought tolerant genotypes screened on the basis of physiochemical and genetic indices can further be used as an effective germplasm while devising a breeding program for drought tolerance adaptability optimization.