Thymus serpyllum L. is an aromatic medicinal herb of temperate and high-altitude regions, yet information on populations from the cold-arid region of Ladakh remains limited. This study examined the phytochemical composition, seed germination behaviour and In vitro propagation potential of T. serpyllum collected from Suru Valley, Kargil. Seed germination was evaluated in a Petri plate bioassay with five pre-sowing treatments using three replicates of ten seeds each. Shade-dried aerial parts were extracted with methanol by Soxhlet extraction and analysed by GC–MS, which revealed 74 chromatographic peaks, of which 25 relatively abundant constituents were examined in detail. For In vitro propagation, nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with six combinations of 6-benzylaminopurine (BAP) and kinetin. GA₃ produced the highest germination (73.3%) compared with 23.3% in the control and in the cold + H₂SO₄ treatment, while cold or H₂SO₄ treatment alone gave no germination. The extract contained a chemically diverse mixture of terpenoid, phenolic, fatty-acid-derived, diterpenoid and sterol-related compounds, including thymol (6.60%), 9,12,15-octadecatrienoic acid (6.11%), 4-vinylbenzene-1,2-diol (5.15%), γ-sitosterol (5.11%), n-hexadecanoic acid (4.67%) and p-cymene-2,5-diol (4.64%). Nodal explants showed only a transient response, with tissue browning preventing sustained shoot proliferation. These findings provide baseline information for the chemical characterisation, seed-based propagation and conservation of T. serpyllum from Suru Valley, and indicate that nodal culture requires further optimisation.