Synchrotron-based infrared (IR) microspectroscopy is able to reveal structural features of biomaterials within intact tissue at both cellular and molecular levels. Heat-related treatments have been used to improve nutrient availability of canola seeds and meal. However, hitherto, there has been no study on the sensitivity and response of each layer in canola seeds to heat-related treatments. It is not known which layer (epiderm/mucllage, spermoderm, endosperm, or cotyledon) is the most sensitive to heat when heat treatment is applied to the seeds. Traditional wet chemical analysis is unable to answer such questions. The objective of this study is to use synchrotron IR microspectroscopy with multivariate molecular spectral analyses as a research tool to study heat treatment effects in a fast way on the structural changes in cotyledon tissues of yellow-type canola (Brassica) seeds among raw (treatment code "A"), wet heating (autoclaving at 121 °C for 60 min, treatment code "B"), and dry heating (dry roasting at 120 °C for 60 min, treatment code "C"). The hypothesis of this study was that different heat treatments have different heat penetration abilities on cotyledon tissues in yellow-type canola seeds. The multivariate analytical tools principal component analysis (PCA) and agglomerative hierarchal cluster analysis (AHCA) were applied to investigate variance and groupings within the spectral data set [whole spectral range of ca. 4000-650 cm(-1), spectral range of ca. 1300-900 cm(-1) (cellulose or saccarides), spectral range of ca. 1800-1500 cm(-1) (secondary structures of protein) and spectral range of ca. 1500-1300 cm(-1) (bending motion of methylene and methyl group; this change is consistent with the change in the range of ca. 3000-2800 cm(-1))]. The results showed that there were no clear cluster and groups formed in the cotyledon tissues among the three treatments (A, B, and C). There were no clear distinguished responses of the cotyledon tissues to different types of heat treatments using multivariate molecular spectral analyses. The results indicate that the cotyledon tissues might not be sufficiently penetrated by both heat treatments (autoclaving and dry roasting) under the specified conditions. A future study is needed to analyze individual functional group band intensity among the treatments using univariate molecular spectral analysis to confirm multivariate PCA and cluster analyses.