Melanocrommyum, a subgenus of the Allium genus, is found in different regions of Iran and is in danger of extinction due to excessive exploitation. This study aimed to determine the genetic diversity in 170 individuals representing 17 wild populations belonging to six endangered species of Allium subg. Melanocrommyum using inter simple sequence repeat (ISSR) markers. The 10 selected ISSR primers produced 178 polymorphic fragments (100%). Polymorphic band number varied from 12 (primer 8) to 22 (primer 2). The average observed number of alleles, effective number of alleles, Shannon's indices and Nei's gene diversity were 1.48, 1.2, 0.2 and 0.1, respectively. According to Nei's genetic distance, the lowest genetic distance (0.048) was observed among both two populations of A. elburzense (Emamzadeh Ebrahim and Kamelat), and two populations of A. subakaka (Jame Shoran and Ghalelan) while the highest distance (0.097) was observed among a population of A. kurdistanicum (Taze Abad Oryeh) with both A. pseudobodeanum (Shen Jari), and A. derderianum (Dareh Oson) populations. In UPGMA cluster analysis, the populations were grouped into four main clusters at a cutoff value of 0.07. The analysis of molecular variance showed that the maximum value of genetic variation was found within the populations (68%), where as a low genetic differentiation was observed among the populations (32%). Our results revealed that ISSR molecular markers are useful to display the diversity in Allium genus and can be used to improve the classification accuracy. This study provided valuable information for the conservation of these species and breeding program planning.