Lepidium sativum L. is a fast-growing, edible and medicinal plant that belongs to the family Brassicaceae. Indian system of medicinal and health (ISHM) recognizes this plant as a source of several medicinal and nutraceutical factors. Ninety-four accessions collected from 19 states of India were assessed for genetic diversity using inter-simple sequence repeat (ISSR) marker. Ten ISSR primers amplified a total of 172 bands across the 94 accessions and out of these, 139 bands were found to be polymorphic and 33 as monomorphic. The percentage polymorphism varied from 60.00 to 91.30% with an average of 80.10%. The polymorphism information content (PIC) varied from 0.14 to 0.39 with an average of 0.27. The Jaccard similarity coefficient ranged from 0.11 to 0.89 with minimum between accession LS61 and LS60 and maximum between accession LS95 and LS81. Cluster analysis based on UPGMA grouped all the 94 accessions into three major clusters with accessions per cluster ranging from 12 to 45. Similar to UPGMA clustering, PCA also differentiated all the accessions into three major groups. Model-based clustering determined three sub-populations (K = 3). Further, analysis of molecular variance showed that 67% of allelic diversity was attributed to individual accessions within populations while 33% was distributed among populations. This preliminary study shows that significant variability exists in the collected accessions.