The objectives of current study was to determine protein characteristics, including crude protein fractionation according to the Cornell Net Carbohydrate and Protein System (CNCPS), in situ ruminal crude protein (CP) degradability and Fourier Transformed Infrared Spectroscopy (FTIR), among barley, corn, and sorghum cultivars. Cereal grains were collected from the central part of Iran and included 1) moderate, cold, and dry climate barley cultivars, 2) early and late maturity corn cultivars and 3) new and conventional sorghum cultivars. The main differences among cereal types were that corn and sorghum contained more starch and less fibre (NDF) than barley (P < 0.05), while soluble CP fractions (PA and PB1), slowly degradable true protein (PB3) and rate and extent of ruminal CP degradation (in situ) were greater (P < 0.05) for barley than corn and sorghum. The FTIR absorbance intensities for amide I, amide II, the ratio of amide I to amide II, α-helix and β-sheets were greater for barley compared with corn and sorghum grain (P < 0.05). Within barley, the moderate type cultivar contained less soluble CP and NPN and more ADICP than the cold and dry type cultivars (P < 0.05), while in situ CP degradation characteristics were similar among cultivars. Protein molecular structures in different cultivars of barley were similar. In corn grain, the early maturing cultivar contained more ash, EE, soluble CP and PB1 and less PB2, PB3 and NDICP than the late maturing cultivar (P < 0.05). Furthermore, FTIR protein molecular structures amide I and II had different between the two corn cultivars. In sorghum, the new cultivar contained less starch, more CP and PB3, and had slower degradation rate (kd) of CP than the old cultivar (P < 0.05). The FTIR protein molecular structures were similar between the sorghum cultivars according to univariate analysis, but cluster and principal component analysis largely separated the FTIR spectra of the two cultivars in the amide region. In conclu