Acid soils limit yields of many important crops including canola (Brassica napus ), Australia's third largest crop. Aluminium (Al3+ ) stress is the main cause of this limitation primarily because the toxic Al3+ present inhibits root growth. Breeding programmes do not target acid-soil tolerance in B. napus because genetic variation and convincing quantitative trait loci have not been reported. We conducted a genome-wide association study (GWAS) using the BnASSYST diversity panel of B. napus genotyped with 35 729 high-quality DArTseq markers. We screened 352 B. napus accessions in hydroponics with and without a toxic concentration of AlCl3 (12μM, pH 4.3) for 12days and measured shoot biomass, root biomass, and root length. By accounting for both population structure and kinship matrices, five significant quantitative trait loci for different measures of resistance were identified using incremental Al3+ resistance indices. Within these quantitative trait locus regions of B. napus , 40 Arabidopsis thaliana gene orthologues were identified, including some previously linked with Al3+ resistance. GWAS analysis indicated that multiple genes are responsible for the natural variation in Al3+ resistance in B. napus . The results provide new genetic resources and markers to enhance that Al3+ resistance of B. napus germplasm via genomic and marker-assisted selection.