Background <p>Alkaline stress poses severe threats to sustainable triticale production. However, lack of molecular and metabolic data restricts the efficient breeding and field management of triticale cultivated in alkaline soils. The aim of this study was to explore the genotypical effects on modifications of transcriptional and metabolic profile in triticale, and find out the prospective genes and metabolites responsible for alkaline tolerance.</p> Results <p>In this study, we compared the root development in two triticale cultivars with contrasting alkali tolerance, and found that root number, length, surface area and biomass were reduced, but the average root diameter was increased in triticale subjected to alkaline stress. The stress effect was extremely significant in the alkali-sensitive cultivar. Comparative transcriptomic and metabolomic analyses revealed that the genotype effect on alkaline stress resistance was predominantly connected with metabolism of amino acids and flavonoids, as well as the featuring biosynthesis of benzoxazinoid and brassinosteroid. Simultaneous regulation of genes and metabolites involved in betalains, dopa and a group of other pathways in the two cultivars were suggested to be basic alkaline stress responses in triticale. Additionally, expression of key genes involved in these processes and typical alkaline stress responses in plants were examined and the subset including <i>AT1</i>-<i>PIP2</i>, <i>RGI1</i>, <i>SAUR215</i>, <i>SCaBP3</i> indicated their implication in alkali tolerance in triticale.</p> Conclusions <p>These gene, metabolite, and pathway resources depict the internal responsive atlas of triticale under alkaline stress. Those involved in the metabolism of key amino acids, flavonoids, and betalains as mentioned above represent prior targets for future genetic studies and breeding of stress-tolerant triticale germplasm to cope with alkaline stress.</p>