The objectives of this study were to investigate the chemical profiles; crude protein (CP) subfractions; ruminal CP degradation characteristics and intestinal digestibility of rumen undegraded protein (RUP); and protein molecular structures using molecular spectroscopy of newly developed yellow-seeded flax (Linum usitatissimum L.). Seeds from two yellow flaxseed breeding lines and two brown flaxseed varieties were evaluated. The yellow-seeded lines had higher (P < 0.001) contents of oil (44.54 vs 41.42% dry matter (DM)) and CP (24.94 vs 20.91% DM) compared to those of the brown-seeded varieties. The CP in yellow seeds contained lower (P < 0.01) contents of true protein subfraction (81.31 vs 92.71% CP) and more (P < 0.001) extensively degraded (70.8 vs 64.9% CP) in rumen resulting in lower (P < 0.001) content of RUP (29.2 vs 35.1% CP) than that in the brown-seeded varieties. However, the total supply of digestible RUP was not significantly different between the two seed types. Regression equations based on protein molecular structural features gave relatively good estimation for the contents of CP (R(2) = 0.87), soluble CP (R(2) = 0.92), RUP (R(2) = 0.97), and intestinal digestibility of RUP (R(2) = 0.71). In conclusion, molecular spectroscopy can be used to rapidly characterize feed protein molecular structures and predict their nutritive value.