Nitrogen (N) fertilization is essential to maximize crop production. However, around half of the applied N is lost to the environment, causing water and air pollution and contributing to climate change. Understanding the natural genetic and metabolic basis underlying plants N use efficiency is of great interest to attain an agriculture with less N demand and thus more sustainable. The study of ammonium (NH4+) nutrition is of particular interest, because it mitigates N losses due to nitrate (NO3–) leaching or denitrification. In this work, we studied Brachypodium distachyon, the model plant for C3 grasses, grown with NH4+ or NO3– supply. We performed gene expression analysis in the root of the B. distachyon reference accession Bd21 and examined the phenotypic variation across 52 natural accessions through analyzing plant growth and a panel of 22 metabolic traits in leaf and root. We found that the adjustment of primary metabolism to NH4+ nutrition is essential for the natural variation of NH4+ tolerance, notably involving NH4+ assimilation and phosphoenolpyruvate carboxylase (PEPC) activity. Additionally, genome-wide association studies (GWAS) indicated several loci associated with B. distachyon growth and metabolic adaptation to NH4+ nutrition. We found that the GDH2 gene was associated with the induction of root glutamate dehydrogenase activity under NH4+ nutrition and that two genes encoding malic enzyme were associated with leaf PEPC activity. Altogether, our work underlines the value of natural variation and the key role of primary metabolism to improve NH4+ tolerance.Read less <