A large panel of Natural Rubber (NR) samples was characterized using Fourier Transform Infrared (FT-IR) spectroscopy in Attenuated Total Reflection (ATR) configuration. Specific vibrational bands were attributed to some non-isoprene compounds naturally present in NR composition. A rapid and non-destructive method was developed to investigate some specific functional groups contained in lipids (ester and carboxyl groups) and proteins (amides). Ester and carboxyl groups were quantified using calibration curves developed from synthetic cis-1,4-polyisoprene mixtures with either methyl stearate or stearic acid. Amide groups of proteins and peptides were found to be directly quantifiable from NR FTIR spectra. The clonal origin and processing were found to influence the non-isoprene composition of NR. Significant correlations were found between FT-IR results and conventional chemical analyses: nitrogen content for proteins and total lipid extract.