Osmotic adjustment is the main component for physiological machinery of wheat drought tolerance. Some of parameters implicated in osmotic adjustment in 15 bread wheat cultivars were evaluated at soil water deficits (50% FC) and FC as the control in the greenhouse. For the physiological traits, analysis of variance showed that there are highly significant differences between treatments, i.e. water stress levels and wheat cultivars. Shahpasand and Marvdasht as sensitive wheat cultivars had significantly lower osmotic adjustment, relative water content, $K^+$ content, soluble sugar, proline, and glycine betaine levels than the rest of the cultivars. The results of cluster analysis revealed that all cultivars were grouped into three distinct clusters. Dez, Kavir, Pishtaz, and Maron cultivars which have the highest osmoregulation activity were in cluster I, whereas clusters II (Pishgam, Aflak, Hirmand, Zagros, and Vee/Nac) and III (Ws-89-2, Sardari, Azar2, Shapasand, and Marvdasht) had intermediate activity and the lowest capacities for osmoregulation, respectively. In addition, in wheat flag leaf during the reproductive stage under drought conditions, the changes in gene expression of two key genes namely P5CS (D-Pyrroline-5-carboxylate synthetase) and BADH (Betaine Aldehyde Dehydrogenase) in two selected cultivars including Dez and Marvdasht revealed that water stress can increase the expression level of the genes P5CS and BADH in the resistant cultivar, Dez, compared with Marvdasht, the sensitive one. In general, it seemed that application of the all cultivars in cluster I would enable breeders to acquire more reliable achievements under drought conditions.