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Genetic diversity among 35 accessions of Ae. cylindrica using 17 ISSR primers were investigated. Totally, 190 alleles were amplified and 188 alleles (98.95%) o were polymorphic. Number of Amplified alleles ranged from 6 to 20 with average 11.18 alleles for each primer. Polymorphic information content (PIC) varied from 0.10 (primer UBC841) to 0.35 (primer UBC836). Marker index criterion ranged from 0.6 (primer UBC841) to 6 (primer 15). Cluster and Principal Coordinate Analysis could not completely separate accessions and showed no association between molecular diversity and geographic diversity of genotypes, indicating that there is high genetic diversity among accessions. West and south-west genotypes showed more diversity than genotypes from north and north-west of the country. Therefore, the center of diversity and origin of Aegilops cylindrica might be the western and south-western regions of country and this species might transferred from these regions to the northern parts of the country. Results of this study showed that ISSR markers are useful tools for management of genetic resources of wheat and their wild relative species.