Girardinia diversifolia (Link) Friis from Urticaceae family is a perennial herb, commonly known as the Himalayan giant nettle. It has cultural, medicinal, and economic values among the indigenous peoples and local communities living in the mountains of Nepal and India. Prior knowledge of genetic diversity in plant species may contribute to conservation and sustainable utilization of important genotype. The aim of this study is to assess the genetic diversity within and among different populations of the 45 accessions of G. diversifolia collected from Far-western, Central, and Eastern regions of Nepal. The amplification of genomic deoxyribonucleic acid (DNA) with ten inter-simple sequence repeat (ISSR) primers yielded 131 clear DNA bands, of which 98.09% were found to be polymorphic. The mean effective number of alleles (ne), Nei's gene diversity (h) and Shannon's information index (I) were 1.598, 0.349, and 0.523, respectively. The total genetic diversity Ht (0.350 ± 0.04), low intrapopulation genetic diversity Hs (0.141 ± 0.03) and low estimated gene flow Nm (0.355 ± 0.11) reflected high genetic differentiation among population Gst (0.594 ± 0.08). The analysis of molecular variance among and within five populations of G. diversifolia showed that the value of genetic variation among the population was 60% whereas the value of genetic variation within the population was 40%. The results show that the ISSR markers are informative for the study of genetic diversity, which may help in the conservation and sustainable utilization of G. diversifolia and allied taxa.