Background <p>White lupin (<i>Lupinus albus</i>, L.) is a grain legume cultivated for its high agronomic and nutritional potential, despite the accumulation of bitter and potentially harmful to health quinolizidine alkaloids (QA) in the grain. Modern sweet (i.e. low alkaloid content) varieties exist, which are exploiting different recessive mutations responsible for the desired low QA chemotype. The most widely used QA-reducing determinant, <i>pauper</i>, has been recently identified enabling marker-assisted selection, but unstable QA content across growing seasons and environments remain a challenge in white lupin cultivation.</p> Results <p>Through Bulked Segregant Analysis of an F₂ population, segregating for two different low QA conferring loci, we identified a novel QTL spanning a 1 Mbp region on chromosome 5, a novel source of sweetness apart from <i>pauper</i>. We present marker-trait associations for the new locus tagging low QA content in white lupin within this QTL, described in F₂ generation and validated in F₃. Together with genotyping of sweetness tagging <i>pauper</i> sweet individuals, we identified, in F₃, 50 stacked allele recombinants where the low QA chemotype is further reduced. These individuals exhibit an exceptionally low total alkaloid content (22.8 ± 10.4 ppm), even when compared to genotypes known to carry the allele associated to the most drastic reduction in QAs, <i>pauper</i> (171.7 ± 18.5 ppm).</p> Conclusion <p>The discovery of this novel locus and the development of associated PACE markers, potentially applicable for marker-assisted selection together with <i>pauper</i> (especially after farther validation in a larger panel of accessions), can enhance the improvement of white lupin through the development of new varieties with very low and stable alkaloid content based on stacked allele recombinants. This can help to increase the cultivation of this useful yet underutilised crop and its use for human nutrition.</p> Graphical abstract <p></p>