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Lepidium sativum (commonly known as garden cress) belongs to the family Brassicaceae. It is a fast-growing erect, annual herbaceous plant. Its seeds possess significant fracture healing, anti-asthmatic, anti-diabetic, hypoglycemic, nephrocurative and nephroprotective activ-ities. In the present study, we assessed the genetic diversity of various genotypes of L. sativum using inter-simple sequence repeat (ISSR) markers. Out of 41 ISSR primers screened, 32 primers showed significant, clear and repro-ducible bands. A total of 510 amplified bands were obtained using 32 ISSR primers, out of which 422 bands were poly-morphic and 88 bands were monomorphic. The percentage of polymorphism was found to be 82. A total of 35 unique alleles ranging insize from 200 to 2,900 bp were observed. Cluster analysis based on unweighted pair-group method, arithmetic mean divided the 18 genotypes into two main clusters, with the first having only HCS-08 genotype of L. sativum and other having all of the other 17 genotypes. The Jaccard similarity coefficient revealed a broad range 32–72%genetic relatedness among the 18 genotypes.