In this study, we developed tissue culture protocol of Salvia dolomitica, a particularly fragrant sage and we evaluated the yield and the profile of essential oil (EO) produced from plants grown in vivo compared to in vitro plant material cultured in two light intensity growth conditions. Plant extracts secreted from glandular trichomes are, in Lamiacea, a valuable source of biologically active volatile compounds. Microscopical observations permitted to describe, for the first time, secretory structures from S. dolomitica leaves developed in vitro showing morphological identity to in vivo glands. The yield of EO obtained from light-stressed in vitro plants was higher than which obtained from the non-stressed in vitro plants and threefold higher that in vivo plants. Phytochemical analysis of EOs and volatile compounds showed quali-quantitative differences between S. dolomitica in vitro grown plantlets and those from open field. α-Pinene, β-phellandrene and borneol accumulated predominantly in micropropagated plants while β-caryophyllene, α-guaiene, δ-cadinene and valencene in in vivo material. Both EO and SPME analysis had the same trend: the percentage of total sesquiterpenes decreased strongly in vitro, on the contrary the total monoterpenes are abundant. The effect of the high light treatment on aromatic profile demonstrating that this stress can affect the EO composition and this can be exploited by researchers to address the accumulation of valuable amounts of EO.