The aim of the work was to characterize the expression of various a-amylase inhibitors (aAIs), well known anti-nutritional compounds, for the development of healthier diploid wheat-based functional foods. The salt-soluble protein fractions from the seeds of 53 accessions among Triticum monococcum subsp. monococcum (T.m.), T. monococcum subsp. boeoticum (T.b.) and Triticum urartu (T.u.) were analyzed by immunoblotting after SDS–PAGE and Urea–PAGE using polyclonal antibodies (PABs) raised against 0.19 and 0.28 aAIs expressed in bread-wheat. Reverse zymography with human saliva and Tenebrio molitor a-amylases was used to assay inhibition activity. A great variability of the expression of aAI-related proteins was observed among T.b. and T.u. PABs, and reverse zymography revealed different bands, often not correlating with those present in bread-wheat. Two-dimensional electrophoresis followed by immunoblotting and mass spectrometric analysis identified these proteins as aAIs. Interestingly, no signal was observed within T.m. accessions. This makes T.m. an important candidate for the production of novel functional foods.