The members of the Aegilops genus serve as a vast pool of allele discovery for wheat improvement in abiotic and biotic stress responses. Aegilops mutica Boiss (Amblyopyrym muticum (Boiss) Eig) is an unexplored candidate with significant potential. Even though it has been used in cytogenetics applications within the last century, natural population diversity and allele discovery have been neglected. As an endemic species for Anatolia and the lower Caucasian region, it has an unexplored population structure. Here, seventy-five genotypes from five different newly collected populations from central Anatolia were evaluated with 29 polymorphic SSR loci. Significant diversity within (83%) and between (17%) the populations was obtained. Three of the populations were clearly separated, while two had some level of the mixture. Relatively easy cross-species hybridization and introgressions make Ae. mutica a good candidate for novel allele discovery and pre-breeding. Here, for the first time, representative natural populations of Ae. mutica were compared and population structures were revealed with SSR markers which may clear the misty vision that geneticists might have regarding Ae. mutica. This could be exploited in genetic resource conservation and breeding programs and maybe a point for further studies.