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Abstract Introduction. The peripheral layers (PL) of wheat, including the aleurone layer (AL), are an important source of micronutrients. AL is excluded, and mainly used for animal feed, although these tissues have significant nutritional potential as they contain most micronutrients, phytochemicals and fiber, and could improve the nutritional quality of foods. Objective. A proteomic approach was used to reveal major protein differences in the aleurone layer (AL) of mature grain between four wheat species: T. Aestivum, and T. spelta (6X), T. durum (4X), and T. monococum (2X). Materiel and methods. In each species, one representative cultivar was used for total AL protein extraction. Two-dimensional electrophoresis of AL proteins revealed 1380, 1355, 1120, and 973 Coomassie stained spots, respectively. Results. A total of 334 spots showed quantitative or qualitative (presence/absence) differences between the four cultivars. T. Aestivum, and T. spelta had 90.9% and 92.6% of their AL proteome, identical, respectively. Only 25 spots were significantly different between the two hexaploid cultivars. AL proteins, encoded by A genome, were mainly expressed in polyploid species. Percentages of proteins, encoded by A genome, was 85%, in tetraploid species, and 70% in the hexaploid spelt. Eighty spots proteins, that are different qualitatively and quantitatively, were identified by mass spectrometry, and data mining, and were classified in 5 biological processes, and 12 functional categories. Conclusion. The major differences between species are, particularly, due to storage globulins (22, 11, and 1 of Glo-3, Glo-3B, Glo-3C, respectively), stress related proteins, including antioxidant proteins, like 1-Cys peroxiredoxin, and manganese superoxide dismutase, defence proteins like xylanase, serpin3, and heat shock proteins.