The main objective of the present study was to standardise the HSI-NIR image acquisition method in naturally contaminated whole wheat kernels to obtain a high accuracy method to quantify and classify samples according to DON levels. To confirm the results, wheat samples were analysed by high performance liquid chromatography as the reference method to determine their DON levels. Hyperspectral images for single kernels and whole samples were obtained and spectral data were processed by multivariate analysis software. The initial work revealed that HSI-NIR was able to overcome kernel orientation, position and pixel selection. The subsequent developed Partial Least Squares (PLS) prediction achieved a RMSEP (Root Mean Square Error of Prediction) of 405 μg/kg and 1174 μg/kg for a cross-validated model and an independent set validated model, respectively. Moreover, the classification ac- curacy obtained by Linear Discriminant Analysis (LDA) was 62.7% for two categories depending on the EU maximum level (1250 μg/kg). Despite of the results are not accurate enough for DON quantification and sample classification, they can be considered a starting point for further improved protocols for DON management.