Key MessageBy integrating QTL mapping, transcriptomics and metabolomics, 138 hub genes were identified in rapeseed root response to aluminum stress and mainly involved in metabolism of lipids, carbohydrates and secondary metabolites.AbstractAluminum (Al) toxicity has become one of the important abiotic stress factors in areas with acid soil, which hinders the absorption of water and nutrients by roots, and consequently retards the growth of crops. A deeper understanding of the stress-response mechanism ofBrassica napusmay allow us to identify the tolerance gene(s) and use this information in breeding-resistant crop varieties. In this study, a population of 138 recombinant inbred lines (RILs) was subjected to aluminum stress, and QTL (quantitative trait locus) mapping was used to preliminarily locate quantitative trait loci related to aluminum stress. Root tissues from seedlings of an aluminum-resistant (R) line and an aluminum-sensitive (S) line from the RIL population were harvested for transcriptome sequencing and metabolome determination. By combining the data on quantitative trait genes (QTGs), differentially expressed genes (DEGs), and differentially accumulated metabolites (DAMs), key candidate genes related to aluminum tolerance in rapeseed were determined. The results showed that there were 3186 QTGs in the RIL population, 14,232 DEGs and 457 DAMs in the comparison betweenRandSlines. Lastly, 138 hub genes were selected to have a strong positive or negative correlation with 30 important metabolites (|R|≥ 0.95). These genes were mainly involved in the metabolism of lipids, carbohydrates and secondary metabolites in response to Al toxicity stress. In summary, this study provides an effective method for screening key genes by combining QTLs, transcriptome sequencing and metabolomic analysis, but also lists key genes for exploring the molecular mechanism of Al tolerance in rapeseed seedling roots.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00122-023-04412-z.