Artemisia annua L. is an important medicinal plant that produces substantial quantity of artemisinin, an antimalarial agent. In India, it grows wild in the Ladhak region and has harboured considerable variability over the years. EST-derived SSR markers were used to measure the genetic diversity among the A. annua germplasm collected from geographically separated Leh (11,500 ft) and Nubra (9600 ft) valleys (Ladakh, India). After analysing 68,974 non-redundant (of 3,60,906 available) ESTs of A. annua, 4,342 SSR markers were developed. On an average, one SSR was found per 8.9 kb of EST sequence with dinucleotide motifs in highest frequency (52.2%), followed by tri (42.4%), tetra (3.6%), hexa (1.2%) and pentanucleotide (0.6%) repeat types. A set of 16 primer pairs were designed by considering only the SSR-containing ESTs from the artetnisinin biosynthetic pathway. In total, 38 alleles were identified from 13 polymorphic SSR loci, ranging from 1-7 alleles per locus and displayed moderate genetic diversity with an average of 0.24. It was found that the genetic diversity among individual from Nubra valley was narrower than that of Leh valley, suggesting the importance and feasibility of introducing elite genotypes from different origins for Artemisia germplasm conservation and breeding programmes.