The Great Gerbil, Rhombomys opimus, is widely distributed in degraded and fragmented desert and semi-desert habitats of the Iranian Plateau. Recent habitat fragmentation from overgrazing by livestock and a government-sponsored rodent control programme threaten to isolate populations and increase their susceptibility to inbreeding and loss of genetic diversity. We examined genetic data of the Great Gerbil in a region where they are data deficient to understand how distance, geography, and anthropogenic factors influence genetic population structure and genetic diversity at the landscape level. We extracted genomic DNA from 109 blood samples collected from nine populations across the Iran’s landscape and amplified four microsatellite loci to examine levels of genetic diversity and genetic population structure. Our analysis revealed two genetic clusters (K=2) that corresponded to R. opimus sodalis in the north slope of the Alborz Mountains (AM) and Kopet-Dagh Mountains (KDM) and R. opimus sargadensis in the south slope of AM and KDM. However, the observed genetic population structure could not be fully explained by north and south slopes of AM and KDM. Genetic variation was low to relatively high (FST ranged from 0.015 to 0.167) and was significant among some populations. We did not find a correlation between genetic distance and geographic distance, which indicates that the geographical distance was not an influential factor in genetic differentiation of the species in Iran. Genetic diversity within the populations studied appears to be the result of a complex mixture of limited local dispersal, social structure favoring female philopatry, and common ancestral frequencies.