A new method for blood detection in whitefish fillets is presented. The method is based on diffuse reflectance hyperspectral imaging in the VIS/NIR range, and unmixing of measured absorbance spectra into known spectra for hemoglobin, water and muscle tissue. Scattering effects are modelled as a polynomial function of wavelength, and are included in the unmixing procedure. The unmixing method is based on a modified Beer-Lambert law and is performed using a non-negativity constrained least squares algorithm. Application of the method to images of samples with varying amounts of blood results in a very good fit between measured and modelled spectra. Reference measurements on homogenized cod muscle with controlled concentrations of added blood are used to model the relationship between the spectral unmixing results and hemoglobin concentration, enabling pixel-by-pixel estimation of hemoglobin concentration. The method is seen as a useful tool for automatic quality grading and processing of fillets in the whitefish industry. (C) 2017 Elsevier Ltd. All rights reserved.