The functionality of anhydrous milk fat (AMF) is determined from solid fat content (SFC) and triacylglycerol (TG) profiles, parameters traditionally measured using nuclear magnetic resonance and high pressure liquid chromatography respectively. Raman spectroscopy coupled with partial least squares (PLS) analysis has been assessed as an alternative method for SFC and TG class quantification. Sample temperature at which the Raman spectra were collected, method of spectral preprocessing and type of PLS analysis were all investigated and found to significantly affect the resulting calibrations (as parameterized by root mean square error of cross validation). Physically heterogeneous AMF samples held at 20 °C were shown to allow reliable SFC predictions on the basis of collected Raman spectra. In contrast to SFC calibrations, physically homogenous samples in a liquid form were ideal for TG class concentration predictions, however, not all TG classes could be reliably predicted.