Stereospecific analysis is an important tool for the characterization of lipid fraction of food matrices, and also of milk samples. The results of a chemical-enzymatic-chromatographic analytical method were elaborated by chemometric procedures such as linear discriminant analysis (LDA) and artificial neural network (ANN). According to the total composition and intrapositional fatty acid distribution in the triacylglycerol (TAG) backbone, the obtained results were able to characterize pure milk samples and milk mixtures with 1, 3, 5% cow milk added to donkey milk. The resulting score was very satisfactory. Totally correct classified samples were obtained when the TAG stereospecific results of all the considered milk mixtures (donkey-cow) were elaborated by LDA and ANN chemometric procedures.