Verbascum alyssifolium is endemic species having considerable narrow distribution in Erzincan (Turkey) Region. Genetic diversity in three natural populations of V. alyssifolium was investigated by ISSR markers. Twenty primers amplified a total of 390 bright and discernible bands of 200-3000 bp, of which 389 bands (PPB=99.74%) were polymorphic in 3 populations. The genetic diversity was high at the populations level; the effective number of alleles (Ne) was 1.4107, and the difference value between observed number of alleles (Na) was 1.9974 and the Nei's genetic diversity (B) was 0.2651, and the Shannon's information index (I) was 0.4206 and a high level of gene flow (Nm = 4.7793) were observed. Analysis of molecular variance showed that 12% of the total genetic diversity resided among populations, while 88% within the populations, in line with low the gene differentiation coefficient (G(sT) = 0.0947). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species.