Nitrogen (N) use efficiency (NUE) in crops is a critical challenge, as only 40 % of applied nitrogen is typically recovered at harvest. Ammonium-based nutrition is proposed as a strategy to increase NUE in agrosystems. However, crops display better performance when growing with a nitrate-based nutrition. To advance in the current understanding of the interspecific variability to ammonium nutrition, we investigated the performance of the three annual species of Brachypodium genus: B. distachyon, B. stacei, and B. hybridum, focusing on their differential responses to ammonium and nitrate nutrition. B. stacei appeared as a tolerant species, with equal growth regardless of the N source, and B. distachyon as the most sensitive, while B. hybridum showed an intermediate phenotype. Metabolomic analysis highlighted critical differences in N metabolism, where B. stacei and B. hybridum exhibited more robust N assimilation in terms of protein content. In addition, several metabolic pathways were found associated with ammonium nutrition. Notably, flavonoid biosynthesis and tricarboxylic acid cycle pathways together with ethylene precursors and iron homeostasis-related compounds contributed to explain species-specific responses to ammonium nutrition. Altogether, these findings pinpoint potential strategies for improving N utilization and ammonium stress tolerance in cereals.Read less <