SSR analysis of rare and endemic species Allium regelianum, which grows in the south of Russia, was performed for the first time. Variability analysis of 88 accessions of A. regelianum was carried out using four highly polymorphic microsatellite loci (PIC value ranged from 0.55 to 0.72). SSR-analysis made it possible to revealed polymorphism within and among the populations of A. regelianum from Volgograd region. Analysis of Wright’s F-statistics and the analysis of molecular variance showed that more than 90% of total genetic variation of the species was attributed to the differences within the populations and less than 10% of the differences were found among the populations. Cluster analysis of 46 accessions of A. regelianum from three populations of Volgograd region and principal coordinate analysis of all accessions did not reveal differentiation among the populations.