Wild white lupins have high levels of alkaloids, which cause a bitter taste, whereas domesticated white lupin varieties have a very low content of alkaloids in seeds. Genes for bitterness from wild white lupins are a contamination threat to domesticated white lupin via cross-pollination. The gene(s) for alkaloid synthesis have not been clearly identified, and the associated molecular background among wild white lupin, domesticated and contaminated domesticated plant materials is unknown. So far, only tigloyl-CoA:(−)-13alpha-hydroxymultiflorine/(+)-13alpha-hydroxylupanine O-tigloyltransferase (HMT/HLTase) cDNA has been cloned based on protein analysis, which was suggested as encoding a quinolizidine alkaloid transferase regulating quinolizidine alkaloid biosynthesis. This gene has not yet been well characterised in important white lupin genotypes. In this study, we found that the majority of the intron sequence of the HMT/HLTase gene differed between wild white lupin accessions P25758 and P27593, and between the commercial varieties. The expression pattern as well as the expression level of the HMT/HLTase gene showed no difference between the P25758 and the low-alkaloid variety Kiev mutant, suggesting the expression of the HMT/HLTase gene has no correlation with bitterness. However, the intron sequence is useful as a DNA marker in the identification of the contamination source of bitter seeds in commercial lupin seed lots.