A well-developed root system benefit host plants by optimizing water absorption and nutrient uptake and thereby increases plant productivity. In this study we have characterized the root transcriptome using RNA-seq and subsequential functional analysis in a set of drought tolerant or susceptible genotypes. The goal of the study was to elucidate and characterize drought-responsive genes in wheat breeding lines that had been through long-term field and biochemical screening of drought tolerance. The results confirm genotype differences in drought tolerance in line with earlier results from field trials. The transcriptomics survey highlighted a total of 14,187 differentially expressed genes (DEGs) that responded to water deficit. The characterization of these genes shows that all chromosomes contribute to drought tolerance, but to different degrees, and the B genome showed higher involvement than the A and D genomes. The DEGs were mainly mapped to flavonoid, phenylpropanoid, and diterpenoid biosynthesis pathways, as well as glutathione metabolism and hormone signaling. Furthermore, extracellular region, apoplast, cell periphery, and external encapsulating structure were the main drought-responsive cellular components in roots. A total of 1,377 DEGs were also predicted to function as transcription factors (TFs) from different families regulating downstream cascades upon drought perception. TFs from the AP2/ERF-ERF, MYB-related, B3, WRKY, Tify and NAC families were the main genotype-specific regulatory factors. To further characterize the dynamic biosynthetic pathways, protein-protein interaction (PPI) networks were constructed using significant KEGG proteins and putative TFs. In PPIs, isozymes from the CYP450, TaABA8OH2, PAL, and GST families play an important role in drought tolerance in connection with MYB13-1, MADS-box and NAC transcription factors. Our findings further amend our current understanding of root transcriptome changes and provide abundant information on target genes and dynamic regulatory networks under drought conditions.