Chromium phytotoxicity results in relevant alterations to plant physiology, gene expression, and genomic DNA methylation at a transgenerational level. Herein, transcriptional responses of durum wheat (Triticum turgidum L.) to chronic chromium exposure were explored in roots and leaves by RNA-seq approach. Plants grown all the time in a hydroponic system supplemented with 2.5 and 10 µM hexavalent chromium were compared to unstressful control plants, assessing biomass and seed yield analyses after senescence. Then, transcriptomic analysis was performed with these plants kept under 10 µM chromium 50 days after the onset of exposure. The chromium concentrations used were considered the lowest dose sufficient to alter gene expression without impeding plant development, while the sampling time reflected the effects in the pre-harvest phase and long-lasting defense mechanisms. Root and leaf samples from plants kept under 10 µM chromium stress and from unstressful control plants were analyzed, generating 12 RNA-seq libraries. In total, 965 and 810 transcripts were found to be differentially expressed, respectively, in roots and leaves in response to 10 µM chromium stress. In roots, transcriptional changes were noted in the primary and secondary metabolism, redox homeostasis, protein modification, solute transport, nutrient uptake, and external stimuli responses. Meanwhile, the transcriptional changes in leaves were primarily found in the secondary metabolism, hormone-related pathways, chromatin modifications, cell division, protein modification and homeostasis, solute transport, and nutrient uptake. In particular, the metal uptake and translocation pathways were studied with greater emphasis to identify key proteins involved in chromium transport and compartmentalization. Furthermore, several genes involved in the biosynthesis of malate-derived organic acids, trace metal transport/detoxification/chelation, and vacuolar compartmentalization were linked to primary defense responses, and some of them were also associated with two putative gene clusters. Therefore, these genes and gene clusters are suggested as valuable biotechnological targets for future proof-of-concept studies aimed at genetic engineering of durum wheat to improve plant tolerance to chromium exposure.