Thymus L. is of great interest in horticulture as ornamentals, spices, and medicinal plants, as well as in the extracts industry due to the richness in bioactive specialized metabolites. The natural hybrid T. × josephi-angeli Mansanet & Aguil. is produced in Spain, as its horticultural forms are very popular for domestic uses and gardening. However, its micropropagation and chemical composition have not been studied yet. Therefore, the main objective of this work was to develop a micropropagation procedure for T. × josephi-angeli, and to check whether the in vitro culture had an impact on the chemical profile of the plants. The results showed a high initiation rate (>91%) after two sterilization treatments were applied. Moreover, a micropropagation rate of around 21 new rooted explants per culture cycle was obtained in treatment M7 (Murashige and Skoog with 0.064 µM 6-(γ,γ-Dimethylallylamino)purine) when compared to the other 10 treatments performed. Acclimatization was successful in all three approaches tested (>75%), and all plants kept growing after 4 months of outdoor cultivation. Finally, 36 volatiles were identified, and the content of major compounds remained not statistically different in acclimatized plants when compared to the wild-type plants according to the analyses made by HS-SPME-GC/MS and SPME-GC/MS. This chemical stability points out the uniformity of the microplants and the suitability of the procedure applied in this study for T.×josephi-angeli horticultural production using in vitro techniques.