ABSTRACT Toxic concentrations of aluminium cations and low phosphorus availability are the main yield-limiting factors in acidic soils, which represent half of the potentially available arable land. Brachiaria grasses, which are commonly sown as a forage in the tropics because of their resilience and low demand for nutrients, have a greater tolerance to high concentrations of aluminium cations than most other grass crops. In this work, we explored the natural variation in tolerance to aluminium cations (Al 3+ ) between high and low tolerant Brachiaria species and characterised their transcriptional differences during stress. We also identified three QTLs associated with root vigour during Al 3+ stress in their hybrid progeny. By integrating these results with a new Brachiaria reference genome, we have identified 30 genes responsible for Al 3+ tolerance in Brachiaria . We also observed differential expression during stress of genes involved in RNA translation, response signalling, cell wall composition and vesicle location genes homologous to aluminium-induced proteins involved in limiting uptake or localizing the toxin. However, there was limited regulation of malate transporters in Brachiaria , which are associated with external tolerance mechanisms to Al 3+ stress in other grasses. The contrasting regulation of RNA translation and response signalling suggests response phasing is critical to Al 3+ tolerance. HIGHLIGHT We identified QTLs, genes and molecular responses in high and low tolerant Brachiaria grasses associated with aspects of response to aluminium stress, such as regulation, cell-wall composition and active transport.