Display omitted •77 volatiles were discovered in analyzed accessions of O. basilicum.•PCA and Cluster analysis were performed based on seven major compounds.•Five chemotypes were identified characterized by one or two dominant constituents. Ocimum basilicum L. exhibits a great variety of cultivars grown for various purposes. The aim of this research was to analyse the chemical composition of 85 O. basilicum accessions cultivated throughout the world using gas chromatography (GC/FID and GC/MS). All accessions were previously categorized into six morphotypes based on 24 morphological traits: True basil, Small-leaf, Lettuce-leaf, Purple basil A, Purple basil B and Purple basil C morphotype. A total of 77 volatiles were observed, with seven of them being in concentrations higher than 10% in at least one accession (1,8-cineole, linalool, linalool acetate, methyl chavicol, eugenol, trans-methyl cinnamate, trans-α-bergamotene). Based on the essential oil composition of 85 O. basilicum accessions, we propose the intraspecific characterization of O. basilicum into five chemotypes: (A) High-linalool, (B) Linalool/trans-α-bergamotene, (C) Linalool/methyl chavicol, (D) Linalool/trans-methyl cinnamate and (E) High-methyl chavicol chemotype. Groups based on morphological and biochemical traits do not necessarily coincide with one another. Due to great morphological and biochemical diversity, both morphological and chemical characterizations are crucial for the effective management of germplasm collections to facilitate the use of the accessions in future breeding programs.