Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils. Studies on Al tolerance have been conducted in Poaceae but their genomes are very complex. Fifty-nine diploid lines (2n=10) of Brachypodium distachyon (L.) P. Beauv. and 37 allotetraploid samples (2n=30) of Brachypodium hybridum Catalán, Joch. Müll., Hasterok & Jenkins sp. nov. were used to evaluate their tolerance to different Al concentrations. B. distachyon is Al-sensitive compared with oat, rice and rye. The diploid lines (except ABR8) were sensitive like barley and Arabidopsis; however, 10 allotetraploid samples were Al-tolerant. Four different root-staining methods were used to detect Al accumulation, cell death, lipid peroxidation and H2O2 production in diploid and allotetraploid plants. The roots treated with Al showed more intense staining in sensitive than tolerant lines. Also, without any staining, the Al treated roots of sensitive plants appear darker than roots from tolerant ones. The study concerning to the organic acids exudation shows that the exudation of citrate and malate was induced only in the roots from tolerant diploid line (ABR8) and tolerant allotetraploid samples. In contrast, the mRNA expression changes of several candidate genes for Al-activated transporters belonging to the ALMT and MATE families were analysed by quantitative PCR (qRT-PCR). The data obtained indicate that the transcripts from BdALMT1, BdMATE1 and BdMATE2 were present mainly in roots and, moreover, that the BdALMT1 transcript is present in higher amounts in the tolerant ABR8 than in the sensitive ABR1 plants indicating that this gene may be involved in Al tolerance. Finally, an insertion was detected in the promoter region of the BdALMT1 of tolerant diploid and allotetraploid plants.