Near-infrared spectroscopy (NIRS) can be a faster and more economical alternative to traditional methods for screening varietal mixtures of nursery plants during the propagation process to ensure varietal purity and to avoid errors in the dispatch batches. The global objective of this work was to develop and optimize a NIR spectral collection method for construction of robust multivariate discrimination models. Three different varieties of Prunus dulcis (Avijor, Guara, and Pentacebas) of agricultural interest were used for this study. Sources of variation were investigated, including the position of the leaves on the trees, differences among trees of the same variety, and differences at the varietal level. Three types of processed samples were investigated. Fresh leaves, dried leaves, and dried leaves in powder form were included in each analysis. A study of spectral pre-treatment methods was also performed, and multivariate methods were applied to analyze the influence of different factors on classification. These included principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and ANOVA simultaneous component analysis (ASCA). The results indicated that variety was the most important factor for classification. The spectral pre-treatment that provided the best results was a combination of standard normal variate (SNV), Savitzky-Golay first derivative, and mean-centering methods. With regard to the type of processed sample, the highest percentages of correct classifications were obtained with fresh and dried powdered leaves at both the training set and test set validation levels. This study represents the first step towards the consolidation of NIRS as a method to identify Prunus dulcis varieties.