Analysis of Triticale (×Triticosecale Wittmack cv. AC Alta) mature pollen proteins quickly released upon hydration was performed using two-dimensional gel electrophoresis followed by mass spectrometry. A total of 17 distinct protein families were identified and these included expansins, profilins, and various enzymes, many of which are pollen allergens. The corresponding genes were obtained and expression studies revealed that the majority of these genes were only expressed in developing anthers and pollen. Some genes including glucanase, glutathione peroxidase, glutaredoxin, and a profilin were found to be widely expressed in different reproductive and vegetative tissues. Group 11 pollen allergens, polygalacturonase, and actin depolymerizing factor were characterized for the first time in the Triticeae. This study represents a distinctive combination of proteomic and molecular analyses of the major cereal pollen proteins released upon hydration and therefore at the forefront of pollen-stigma interactions.