COTONEASTER orbicularis Schltdl, native to Egypt and the eastern part of the Arab peninsula, is an endangered species and the surviving individuals are dispersed in the mountains of South Sinai, Egypt. Several threats, including habitat aridification and the impact of human interference have caused a noticeable reduction in population size and number. Analyzing the genetic diversity and mating system of C. orbicularis is crucial for its conservation and management. In this study, six polymorphic microsatellite markers, formerly developed as species-specific markers, were used to assess the genetic variability and extent of inbreeding in the C. orbicularis populations. Flow cytometry of seeds was used to establish genome size. Low total heterozygosity (HT = 0.464) for all loci and populations, high differentiation between populations (FST = 0.634, RST = 0.889), and high levels of inbreeding (F = 0.906) were found. Most of the embryos possessed about 1.9 pg/2C DNA; however, embryos with 3.8 pg/2C DNA also occurred. The results provide markers that can be used in the management of conservation, including habitat recovery and ex-situ conservation, in order to prevent extinction.