Flaxseed (Linum usitatissimum L.), also known as linseed, is one of the most important commercial oilseed crop of Western Canada, providing valuable inputs for food, feed, manufacturing and pharmaceutical industries. The objectives of this study were to investigate chemical profiles; protein subfractions; ruminal CP degradation and intestinal digestibility of RUP of flaxseed in dairy cattle; and protein molecular structures of newly developed yellow-seeded flax. Four flaxseed genotypes with 2 yellow breeding lines and 2 brown varieties obtained from Crop Development Center were evaluated. Each genotype were sampled from 2 replicate plots at 2 different locations. The flaxseed samples were analyzed for protein molecular profiles in terms of amide I and II, α-helix and β sheet structures using a JASCO FT/IR-ATR. The rumen and intestinal digestion of flaxseed were determined using in situ and 3-step in vitro methods with 4 lactating Holstein dairy cows (body weight, 680 ± 10 kg). The cows were fed TMR with forage to concentrate ratio 50 to 50. The data were analyzed as a RCBD with in situ cows as a random effect using the PROC MIXED procedure of SAS. The results showed that the yellow-seeded lines had higher (P < 0.001) contents of oil (44.5 vs. 41.4% DM, SEM = 0.35) and CP (24.9 vs. 20.9% DM, SEM = 0.96) compared with the brown-seeded varieties. The CP in yellow-seeds contained lower (P < 0.01) contents of true protein subfraction (81.3 vs. 92.7% CP, SEM = 1.46), and more (P < 0.001) extensively degraded (70.8 vs. 64.9% CP, SEM = 0.46) in rumen resulting in lower (P < 0.001) content of RUP (29.2 vs. 35.1% CP, SEM = 0.71) than the brown-seeded varieties. However, the total supply of digestible RUP was not significantly different between the 2 seed types. Regression equations based on protein molecular structural features gave relatively good estimation for the contents of CP (R² = 0.87), soluble CP (R² = 0.92), RUP (R² = 0.97) and intestinal digestibility of RUP in dairy cattle (R² = 0.71). In conclusion, molecular spectroscopy can be used to rapidly characterize flaxseed protein molecular structures and predict their nutritive value in dairy cattle.