<jats:title>Abstract</jats:title><jats:p>This article presents a novel and original approach to analyze in situ the main components of <jats:italic>Eucalyptus</jats:italic> oil by means of Raman spectroscopy. The obtained two‐dimensional Raman maps demonstrate a unique possibility to study the essential oil distribution in the intact plant tissue. Additionally, Fourier Transform (FT)‐Raman and attenuated total reflection (ATR)‐IR spectra of essential oils isolated from several <jats:italic>Eucalyptus</jats:italic> species by hydrodistillation are presented. Density Functional Theory (DFT) calculations were performed in order to interpret the spectra of the essential oils of the <jats:italic>Eucalyptus</jats:italic> species. It is shown that the main components of the essential oils can be recognized by both vibrational spectroscopic techniques using the spectral information of the pure terpenoids. Spectroscopic analysis is based on the key bands of the individual volatile substances and therefore allows one to discriminate different essential oil profiles of several <jats:italic>Eucalyptus</jats:italic> species. It has been found that the presented spectroscopic data correlate very well with those obtained by gas chromatography (GC) analysis. All these investigations are helpful tools to generate a fast and easy method to control the quality of the essential oils with vibrational spectroscopic techniques in combination with DFT calculations. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 237–248, 2005