Hesperozygis myrtoides is a native species from Cerrado and Atlantic Forest of Brazil. The essential oil from aerial parts of this plant, collected at different periods of the year, was obtained by hydrodistillation and analyzed by GC and CG-MS. The most abundant compounds were limonene (2.1â22.7%), isomenthone (14.3â47.7%), neo-isomenthol (1.8â4.4%), pulegone (19.8â57.3%) and isomenthyl acetate (0.3â14.3%). The chromatographic data were processed using hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on the major compounds. The multivariate analyses (MVA) indicated that the variation of chemical composition of principal substances of H. myrtoides essential oil depends on the altitude. The enantiomeric distribution of (R)-(+)-pulegone and (+)-isomenthone were established for the first time for this essential oil by bidimensional GC-chiral analysis.