The aleurone layer (AL) is an inner tissue of the wheat grain. It contains micronutrients, vitamins, antioxidants and fibre, and can greatly increase the nutritional quality of flour if it is not removed from the kernel with the bran. The AL of mature kernels of three varieties of the two major cultivated wheat species T. aestivum (genome A, B and D) and T. monococcum (genome A) were manually dissected and analyzed using two-dimensional gel-based proteomics. In T. monococcum although composed of only genome A, the maximum number of Coomassie stained AL spots was close to the number found in the bread wheat varieties (1320 and 1258, respectively). Inter-variety variation in spots was higher in the three T. monococcum varieties (103 spots) than in the three T. aestivum varieties (79 spots). Comparison of the two species revealed that only 88 spots differed significantly either in abundance or presence/absence. The B and D genome did not drastically modify the AL proteome, as demonstrated by the fact that 93% of the spots present in T. Monococcum AL spots were also present in T. aestivum. Proteins which differed within and between species were identified using MALDI-TOF and LC-MS/MS Mass Spectrometry. Among the 182 spots that differed, 115 were identified, 53 differed between the two species and 44 (83%) were globulin (Glo) storage proteins. The remarkable environmental stability of the AL proteome previously observed in T. durum and T. aestivum species was confirmed in the variety T. monococcum DV92, grown for two consecutive years in field conditions. Only 15 proteins (out of 1320 AL spots) exhibited significant quantitative variations.