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 between high and low tolerant Brachiaria species and characterised their transcriptional differences during stress. We also identified three QTLs associated with root vigour during aluminium cation stress in their hybrid progeny. By integrating these results with a new Brachiaria annotated reference genome, we have identified genes associated with aluminium cation tolerance in Brachiaria. We observed differential expression of response signalling, cell wall composition and vesicle transport genes homologous to aluminium-induced proteins involved in limiting uptake or localising toxic agents. There was also evidence in Brachiaria of external tolerance mechanisms, such as regulation of malate transporters, common in other grasses. Contrasting regulation during Al3+ stress of numerous genes involved in RNA translation suggests variations in response timing may be responsible for the differences in tolerance to aluminium cations among Brachiaria species.