Abiotic stress conditions cause extensive losses to agricultural production worldwide. Salinity and alkalinity affect plant growth, photosynthesis and availability of nutrients including Fe. Many studies have described the response mechanisms of plants to single abiotic stress conditions. However, in the field, crops and other plants are routinely subjected to a combination of different abiotic stresses. Salinity and alkalinity are wide-spread in Tunisia, where Medicago truncatula occurs as a native species. We established a growth system to study the combined effects of salinity and alkalinity conditions in laboratory conditions. We screened 11 Tunisian M. truncatula lines from the SARDI collection based on their phenotypic responses to the double stress. Salinity and alkalinity affected germination rates, shoot and root dry weights, pigment contents and root morphology parameters. We were able to select among the 11 investigated lines four sensitive and tolerant lines with different abilities to respond to the double stress. Tolerant and sensitive genotypes (two lines each) differed in root flavin contents, root flavin staining patterns and concentrations of root flavins in the nutrient solution. Root architecture, flavin root localization in epidermal cells and flavin secretion are relevant tolerance mechanisms for salt and alkaline stress in M. truncatula. Pairs of contrasting lines from close origins were identified that will be useful tools to identify genes for the tolerance mechanisms.