<p>Cuticular hydrocarbons (CHCs) protect insects from dehydration and pathogen attack and participate in interindividual communication. In social insects, the CHC profile serves as a chemical identification system that communicates information on nest membership, life stage, caste, and fertility. It is known that factors such as diet composition, temperature, and humidity may affect the cuticular profile of leaf-cutting ants under laboratory conditions, hindering intercolony recognition. This study aimed to determine whether the CHC profile of leaf-cutting ants under natural conditions is influenced by the dominant type of vegetation. <i>Atta sexdens</i> (Linnaeus,1758) foraging workers were collected at different times of the year from nests located in a eucalyptus plantation and an Atlantic Forest site. Cuticular extracts were analyzed by gas chromatography–mass spectrometry. The CHC profiles of ants collected from both sites were compared qualitatively and quantitatively by discriminant analysis. In total, 17 compounds were detected in <i>A. sexdens</i> cuticles (<i>n</i>-C16 to <i>n</i>-C31), including 15 compounds in ants from the eucalyptus plantation and 13 compounds in ants from the Atlantic Forest. Of the 17 compounds, 8 were methylated CHCs, 3 were linear alkanes, 3 were unsaturated hydrocarbons, and 3 could not be identified. Ants collected from different sites contained unique cuticular compounds but did not differ in the relative concentrations of common compounds. Qualitative differences in CHC profile between ants inhabiting different environments can be explained by differences in diet composition. Furthermore, quantitative variations in CHCs throughout the year can be partially attributed to physiological responses to seasonal climatic conditions.</p>