AbstractThere is an urgent need for the improvement of drought-tolerant bread and durum wheat. The huge and complex genome of bread wheat (BBAADD genome) stands as a vital obstruction for understanding the molecular mechanism underlying drought tolerance. However, tetraploid wheat (Triticum turgidum ssp., BBAA genome) is an ancestor of modern bread wheat and offers an important model for studying the drought response due to its less complex genome. Additionally, several wild relatives of tetraploid wheat have already shown a significant drought tolerance. We sequenced root transcriptome of three tetraploid wheat varieties with varying stress tolerance profiles, and built differential expression library of their transcripts under control and drought conditions. More than 5,000 differentially expressed transcripts were identified from each genotype. Functional characterization of transcripts specific to drought-tolerant genotype, revealed their association with osmolytes production and secondary metabolite pathways. Comparative analysis of differentially expressed genes and their non-coding RNA partners, long noncoding RNAs and microRNAs, provided valuable insight to gene expression regulation in response to drought stress. LncRNAs as well as coding transcripts share similar structural features in different tetraploid species; yet, lncRNAs slightly differ from coding transcripts. Several miRNA-lncRNA target pairs were detected as differentially expressed in drought stress. Overall, this study suggested an important pool of transcripts where their manipulations confer a better performance of wheat varieties under drought stress.