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Durum wheat (Triticum turgidum L. ssp. durum) landraces, traditional local varieties representing an intermediate stage in domestication, are gaining attention due to their high genetic variability and performance in challenging environments. While major kernel metabolites have been examined, limited research has been conducted on minor bioactive components like lipids, despite their nutritional benefits. To address this, we analyzed twenty-two tetraploid accessions, comprising modern elite cultivars and landraces, to (i) verify if the selection process for yield-related traits carried out during the Green Revolution has influenced lipid amount and composition; (ii) uncover the extent of lipid compositional variability, giving evidence that lipid fingerprinting effectively identifies evolutionary signatures; and (iii) identify genotypes interesting for breeding programs to improve yield and nutrition. Interestingly, total fat did not correlate with kernel weight, indicating lipid composition as a promising trait for selection. Tri- and di-acylglycerol were the major lipid components along with free fatty acids, and their relative content varied significantly among genotypes. In particular, landraces belonging to T. turanicum and carthlicum ecotypes differed significantly in total lipid and fatty acid profiles. Our findings provide evidence that landraces can be a genetically relevant source of lipid variability, with potential to be exploited for improving wheat nutritional quality. [ABSTRACT FROM AUTHOR]