Abstract Linum glaucum is a member of the section Linum of the genus Linum. This species is distributed in the western regions of Iran. In the present study, the inter-simple sequence repeat (ISSR) technique was used to estimate infraspecific variation in different populations of L. glaucum. Samples from nine populations were collected from different regions of the country during spring 2011. For ISSR investigations, populations were divided into three geographical regions. Detrended correspondence analysis showed that ISSR loci were distributed in different parts of the genome and showed no clustering with close association. Gst analysis of ISSR loci showed that some of these loci have good discriminating power. STRUCTURE analysis showed that except for population No. 9, the other populations contained almost similar allelic composition, but differed in the frequency of these alleles using ISSR data. The studied populations were separated in principal coordinate analysis plots and in unweighted paired group cluster analysis. Based on the observed variation, populations were distinguished as ecotypes in the studied populations.