Intra-species genetic variability assessment is an effective tool in formulating genetic improvement and germplasm conservation strategies. Houttuynia cordata Thunb. is a semidomesticated medicinal herb consumed widely in traditional diet in North-Eastern India. In the present study, an effort has been made to assess the genetic diversity of H. cordata Thunb. from Brahmaputra valley of North-East India. A total of 545 genotypes from 18 populations of H. cordata Thunb. from four different regions, i.e. North-East, North-West, South-East and South-West, with respect to river Brahmaputra were collected and population genetic diversity and structure were analysed using ISSR molecular markers. Population genetic structure analysis using unweighted pair group method with averages (UPGMA)-based hierarchical cluster analysis, principal coordinate analysis (PCoA) and model-based clustering in STRUCTURE program revealed that the population of H. cordata Thunb. grouped according to regional distribution and forms four genetically distinct clusters. The analysis of molecular variance showed that differentiation among regions was significant with 60% genetic variation among region, 3% genetic variation among population within region and 37% genetic variation within population. We found wide variation in Nei’s gene diversity (Hj) ranging from 0.07782 in Margherita population to 0.13634 in Barapani population. Furthermore, Nei’s gene diversity within population (Nei’s Hs) and total gene diversity (Ht) were found to be 0.1081 and 0.1769 respectively. The genetic differentiation among 18 population was high (Fst = 0.3894; p < 0.001) with relatively restricted gene flow (Nm = 0.6564). Based on the result of this study, we suggest ex situ conservation could be an appropriate measure to adequately capture the total genetic diversity of H. cordata Thunb. populations of North-East India by selecting few individuals from different populations.