Astragalus argaeus is critically endangered endemic species growing only on Erciyes Mountain in Kayseri. Inter simple sequence repeat (ISSR) markers were chosen to detect the genetic diversity in four populations of A. argaeus. Ten primers were used to assess the diversity among 96 genotypes collected from the four localities in Erciyes Mountain. A total of 78 bands were scored, of which 44 (55.8%) were polymorphic. The unweighted pair group method arithmetic average (UPGMA) and principle component analysis (PCoA) showed moderate genetic diversity at the species and population level. The percentages of polymorphic bands (PPB) ranged from 53.8 to 61.5 (58.01% ± 3.2) and average gene diversity (h) at the population and species level was estimated to be 0.17 and 0.23, respectively. The Shannon’s information index (SI) ranged from 0.24 to 0.29 at the population level and was 0.35 at the species level. The determined gene flow (Nm) was 1.83. The UPGMA tree indicated that the four populations were not genetically distinct obviously. In analysis of molecular variance (AMOVA), the percentage of the variance was 38.72% among populations and 61.28% within populations. The data which small population size, habitat fragmentation and moderate levels of genetic diversity demonstrate that A. argaeus possess threat of extinction if its narrow habitat is destroyed.