Gebel Mousa located in Saint Katherine Protectorate (SKP), South Sinai, Egypt. It harbors many endemic and endangered medicinal plants that are overexploited and facing serious threat of extinction. From these important medicinally plant species, we select Thymus decussatus (near-endemic and endangered plant) to investigate the genetic diversity level among eight populations using both of random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Seven primers were chosen for RAPD and six primers for ISSR analysis. Thirteen RAPD and ISSR primers amplified 97 bands with an average of 7.46 fragments / primer and the total number of polymorphic fragments was 84, thus, representing a level of polymorphism of 86.70%. Jaccard's similarity coefficient values ranged from 0.37 to 0.64. The highest similarly value (0.64) was scored between Farsh Shoeib and Corridor between Farsh El-Hamar & Farsh Shoeib populations as the closet. On the other hand, the lowest similarity value (0.37) was recorded between Farsh Ellya in direction of G. Mousa and Corridor between Farsh El-Loza & Farsh Ellya populations as most distant. The dendrogram separated the eight T. decussates populations into two clusters. The first cluster included Farsh El-Sefsafa, Farsh El-Hamar; Corridor between Farsh El-Loza & Farsh Ellya. The second cluster included Farsh Shoeib, Corridor between Farsh El-Hamar & Farsh Shoeib, Farsh Ellya and Farsh Ellya in direction of G. Mousa and Farsh El-Loza. Assessing of genetic polymorphisms among the Thymus populations at varying microhabitats can help in their genetic improvement and their conserving programs.