Near-infrared reflectance (NIR) spectroscopy was used in the characterization of grain morphology mutants of barley ( Hordeum vulgare L.) in relation to grain nitrogen (N) content and protein composition. Derivative spectroscopy provided spectra with enhanced resolution, allowing wavelengths to be identified with clear differences in contribution from associated chemical bonds. Comparisons of fourth-derivative spectra of wholemeal flour from high-N grains with flour from low-N grains identified wavelengths at which there were statistically significant differences between the groups. Their importance was independently confirmed by step-up regression using these wavelengths to generate an equation predicting N content (R(2) = 0.98). Fourth-derivative spectral comparisons also allowed novel biochemical differences to be predicted. Visual assessment of the spectra of all mutants revealed a variable region (1470-1520 nm, corresponding to N-H stretch vibrations) that allowed two extreme sets to be defined. The protein extracted from these two sets differed markedly in hordein content.