The aim of this study was to determine the differences between two Grapholita molesta (Busk) populations by applying infrared spectroscopy. Grapholita molesta populations were collected from different regions within Porto Amazonas, Brazil, and Emilia Romagna, Italy. Eggs, fifth instars, pupae, and adults were examined. Pupae and adults were sexed. A spectrophotometer that was set between 400 and 4000 cm(-1) range was used to perform 32 scans. The spectra obtained were analyzed using partial least squares (PLS), and external validation of the model was conducted with Unscrambler(TM) software, Version 9.1 (CAMO Software AS, Oslo, Norway). The results showed the differences between the populations in all of the analyzed stages of maturation. The pupal stage was the most viable stage for reading the beam spectrum, and it was concluded that IR can separate G. molesta biotypes.