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Picrorhiza kurroa (family Scrophulariaceae) is a high value medicinal herb used in herbal drug formulations like picroliv, picrolax, etc. It has been collected recklessly from its natural habitat posing endangered status for its existence, thereby necessitating characterization of genetic diversity for its sustainable utilization and conservation. In this study, picrosides content and genetic profiles of 26 accessions of P. kurroa from different locations in north-western Himalayas have been analysed. Picroside-I (P-I) content ranged from 0.37 to 2.7% in fresh shoots whereas total picrosides content (P-I+P-II) ranged from 3.7 to 10.9% in dry rhizomes. High picrosides content accession PKS-1 was identified, both for shoots and rhizomes. To study genetic diversity and correlate picrosides content with their genetic factors, genetic profiling was done using simple sequence repeats (SSRs) identified from P. kurroa transcriptomes. Out of 361 SSR primers tested on 26 accessions, 35 primers yielded polymorphic profiles. Overall low genetic diversity was observed in P. kurroa accessions. The highest polymorphism information content (PIC) of 0.55 was given by SSR marker PKSTS-P9 (TGGTG)4. Mean allele number was 2.97. Mean observed and expected heterozygosities were 0.597 and 0.452, respectively. Among all accessions Nei's genetic diversity (H) was 0.39 and Shannon's information index (I) was 0.58. Cluster analysis of STRUCTURE was comparable to DARWIN. Microsatellite markers would be helpful in the development of DNA diagnostics for the authentication of quality plant material as well as planning a genetic improvement strategy. [ABSTRACT FROM PUBLISHER]