The Saker Falcon is a valuable and endangered raptor species whose populations have sharply declined due to habitat destruction, illegal hunting, trafficking, and human disturbances. Owing to its geographical position and habitat diversity, Iran represents one of the important regions within this species’ distribution range; however, no information has been available regarding the genetic structure of its populations in the country. This study aimed to conduct an initial assessment of the genetic diversity and structure of Saker Falcons in Iran using microsatellite markers. Despite extensive efforts to collect samples nationwide, only 10 individuals were obtained from Tehran, Bushehr, Kerman, and North Khorasan provinces, sourced from the tissue bank of the National Museum of Natural History and Genetic Resources of Iran's Department of Environment. Based on recent studies, eight microsatellite loci appropriate for this species were selected for this study. The results showed that among the eight loci examined, only one locus (SSR57) exhibited significant evidence of null alleles. Likewise, the Hardy–Weinberg equilibrium test indicated a significant deviation only in this locus. All loci used in this study displayed high polymorphism, with eight to twelve alleles per locus. Furthermore, the observed heterozygosity ranged from 0.6 to 1.0, while the expected heterozygosity ranged from 0.794 to 0.939, indicating relatively high genetic diversity in Iranian Saker Falcon populations. The overall inbreeding coefficient (FIS) was estimated at 0.084, suggesting limited inbreeding among the analyzed samples and implying that, despite existing threats, Iranian populations of the Saker Falcon have retained an adequate level of genetic diversity. Additionally, bottleneck analysis provided no evidence of a recent considerable population decline in Iran. Population structure analyses based on Bayesian inference and maximum-likelihood approaches indicated that the samples were grouped into two distinct genetic clusters. Given that two subspecies, F. c. cherrug and F. c. milvipes, have been reported in Iran, these two identified genetic clusters may confirm the presence of both subspecies within the country. This study provides a preliminary insight into the status of genetic diversity and population structure of the Saker Falcon in Iran. Further studies based on sampling from all regions within the distribution range of this species in Iran are necessary.