AbstractGround level UV-B (290–315 nm) and UV-A (315–400 nm) radiation regulates multiple aspects of plant growth and development. In a natural environment, UV radiation interacts in a complex manner with other environmental factors (e.g., drought) to regulate plants’ morphology, physiology, and growth. To assess the interactive effects of UV radiation and soil drying on plants’ secondary metabolites and transcript abundance, we performed a field experiment using two different accessions of Medicago truncatula (F83005-5 French origin and Jemalong A17 Australian origin). Plants were grown for 37 days under long-pass filters to assess the effects of UV short wavelength (290–350 nm, UV_sw) and UV-A long wavelength (350–400 nm, UV-A_lw). Soil–water deficit was induced by not watering half of the plants during the last seven days of the experiment. The two accessions differed in the concentration of flavonoids in the leaf epidermis and in the whole leaf: F83005-5 had higher concentration than Jemalong A17. They also differed in the composition of the flavonoids: a greater number of apigenin derivatives than tricin derivatives in Jemalong A17 and the opposite in F83005-5. Furthermore, UV_sw and soil drying interacted positively to regulate the biosynthesis of flavonoids in Jemalong A17 through an increase in transcript abundance of CHALCONE SYNTHASE (CHS). However, in F83005-5, this enhanced CHS transcript abundance was not detected. Taken together the observed metabolite and gene transcript responses suggest differences in mechanisms for acclimation and stress tolerance between the accessions. Graphical abstract