There is a continual need for development of rapid methods that meet or exceed the detection levels of currently available analytical methods for authentication of food products. The objective of this study was to evaluate temperature-controlled attenuated total reflectance-mid-infrared (ATR-MIR) spectroscopy combined with multivariate analysis as a simple and rapid method for the determination of butter adulteration as a dairy food system. Commercial samples of butter fat were adulterated with margarine fat at levels ranging from 0% to 100% (v/v). Partial least square regression (PLSR) models gave standard error of cross-validation (SECV) of <1.2% (v/v) and correlation coefficients (r) > 0.99. Excellent predicting capabilities were obtained using an external validation set consisting of butter adulterated with margarines at ratios of 2.5%, 13%, and 45%. We have demonstrated the feasibility of a temperature-controlled ATR-MIR spectroscopy technique that would allow for rapid analysis of dairy products. © 2010 Elsevier Ltd. All rights reserved.