The FT-IR spectra of L. amazonensis, L. chagasi, and L. major were analyzed by multivariate statistical analysis (cluster analysis) and the deconvolution procedure based on spectra second derivatives. The cluster analysis demonstrated heterogeneity of 500 when comparing L. amazonensis, L. chagasi, and L. major at the same time; and a heterogeneity of approximately 60 when comparing L. chagasi and L. major. To elucidate the biochemical basis of these differences, the FT-IR spectra of L. amazonensis, L. chagasi, and L. major was analyzed by Gaussian deconvolution. The parasites were discriminated by several spectral bands, attributed to differences in the amount and polymorphism of polysaccharide, fatty acids (phospholipids), nucleic acid, and proteins (amide III).