Kochia scoparia (L.) Schrad is a wild species distributed over Iran especially in Borujerd with medicinal and ecological values. To conserve its breeding and germplasm resources, fifteen DNA-based molecular ISSR markers were used for genetic diversity evaluations of 334 samples from fourteen different natural habitats, including in this study. DNA extraction from young and fresh leaves of plants was isolated using CTAB technique. A total of fifteen ISSR primers in all population produced of reproducible 28114 bands produced, 16989 of them were polymorphic (60.42%). The total length of bands created between 85 to 1660 bp was variable. Matrix 1/0 bands ISSR by MVSP Ver 3.2 and NTSYSpc Ver 2.02 clustering UPGMA of Jaccard coefficient and similar groupings were obvious in the PCoA analysis. The results showed the presence of seven separated genetic group among studied populations in Borujerd. The highest genetic similarity coefficient (0.92) was observed between Bs13 and Bs14 and lowest similarity was between Bs6 and Bs11 (0.25). There was no significant relationship observed between the genetic distance of populations and their geographic distribution. This study indicates the molecular markers ISSR could characterize a wide range of genetic diversity and were able to group genotypes well. Furthermore, understanding such genetic diversity is necessary and useful in the management of Kochia scoparia (L.) Schrad germplasms.