Root-zone temperature affects several physiological processes, but its influence on alkaloid biosynthesis and accumulation in plants has not yet been evaluated. Here we studied the effect of different root-zone temperatures on Catharanthus roseus and Nicotiana tabacum, which produce a variety of alkaloids. C. roseus produces different alkaloids in different organs and tissues. N. tabacum produces its main alkaloid, nicotine, only in the roots. Plants were maintained for two days at root-zone temperatures of 12 °C, 25 °C (control temperature), and 30 °C; the alkaloid content of the roots and shoots of the plants was then analysed, and the expression level of genes involved in alkaloid biosynthesis pathways was determined. In C. roseus, the lowest vinblastine content was detected in the leaves of plants with a root-zone temperature of 30 °C. Incubation at a root-zone temperature of 12 °C greatly enhanced the root ajmalicine content. In general, the expression of genes involved in alkaloid biosynthesis was highest in C. roseus plants with a root-zone temperature at 12 °C. In N. tabacum plants, a root-zone temperature of 30 °C reduced the root nicotine content by 50% compared to controls, while the anabasine content was reduced by approximately 30% in plants with a root-zone temperature of 12 °C or 30 °C compared to control plants. Genes involved in the nicotine pathway were more highly expressed in plants with a root-zone temperature of 12 °C. Our results demonstrate that the root-zone temperature alters the biosynthesis and accumulation of alkaloids in plant tissues. Our results also suggest that cross-talk between the roots and shoots mediates the transcription of genes involved in alkaloid biosynthesis in different parts of the plant.