Hemidesmus indicus (L.) R.Br. is a highly valued medicinal herb in both ancient and contemporary Indian medicinal systems. However, the species has recently become vulnerable due to indiscriminate collection and the consequent rapid decline of its natural populations. Therefore, assessing its genetic diversity is essential to support future conservation and restoration initiatives. In the present study, genetic diversity was evaluated across five natural populations of H. indicus distributed across the north-eastern states of India using 12 inter-simple sequence repeat (ISSR) primers. A total of 54 DNA fragments were generated, of which 27 (50 %) were polymorphic. The polymorphic information content (PIC) ranged from 0.24–0.55, with a mean value of 0.43. The number of observed alleles (Na) varied from 1.16–1.25, the effective number of alleles (Ne) from 1.12–1.20 and Shannon’s information index (I) from 0.10–0.16. Nei’s genetic differentiation index (Gst = 0.437) indicated moderate genetic differentiation among populations, while analysis of molecular variance (AMOVA) revealed that 73 % of the total genetic variation was distributed within populations. Cluster analysis based on Jaccard’s similarity coefficient separated the five populations into two major groups, which was further supported by the results of principal component analysis (PCA). The present findings provide baseline information on the genetic structure of H. indicus in Northeast India and demonstrate the effectiveness of ISSR markers in assessing genetic variation. These results are valuable for formulating conservation strategies, germplasm management and future improvement and domestication programmes of this important medicinal species.