The mid- and near-infrared (mid-IR and NIR) spectra of aqueous solutions of glucose and fructose, fructose and galactose, and glucose and galactose were recorded and analyzed by heterospectral two-dimensional correlation spectroscopy (H2D-CS) to determine characteristic NIR wavelengths for each sugar. Fourier self-deconvolution (FSD) was applied to the NIR spectra prior to H2D-CS analysis to help resolve the strongly overlapping sugar absorptions. Examination of the H2D-CS data gave characteristic absorption wavelengths for glucose, fructose, and galactose. The wavelengths identified by H2D-CS were then used to develop multiple linear regression (MLR) calibrations for the quantitative analysis of mixtures of the three sugars in solution. This approach gave comparable results to MLR calibrations based on wavelengths selected by examination of the first- and second-derivative spectra of solutions of the individual sugars.