Legumes produce protein-rich seeds and have been cultivated for centuries to feed humanity and livestock. Despite their ability to fix dinitrogen, seedlings are sensitive to nitrogen availability before symbiosis establishment. In the past years, our team has been working on seedling pre-emergence (i.e. heterotrophic) growth with and without nitrogen source, using a broad panel of accessions of barrel medic (Medicago truncatula), a model legume plant. In other Legumes like pea, sulfur metabolism appears to be critical during germinative and post-germinative events. Thus, we assume sulfur nutrition may influence seedling growth properties of genotypes. The present study focuses on the genotypic variability on a selection of accessions of M. truncatula during the heterotrophic growth, under different nutrient conditions, including sulfate availability. We determined traits associated with seedling performance (seed biomass use and organ elongation) to characterize phenotypic variations in response to contrasting nitrate and sulfate conditions. The study reveals that genotype response to nitrate deficiency is influenced by sulfate availability, the genotype × nutrient conditions interaction being more or less pronounced depending on the trait. Elemental, isotopic and inorganic anion analyses of seeds and seedling organs also reveal genetic variability and highlight genotype x nutrient conditions interaction, suggesting differential N/S metabolic interactions amongst accessions.