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Substantial variation in phenotypic traits and ISSR fingerprinting was observed among twenty populations of Achillea fragrantissima (Forssk.) Sch. Bip. in Egypt. Such variation was reflected in the clustering of the examined populations into two major groups: one representing populations in the mountainous area of South Sinai and the other comprising populations growing at low elevations in the middle of Sinai and the desert west of the Suez Canal from Suez in the east to Cairo in the west. Five populations in the eastern part of Sinai near Nuwieba and Taba on the Gulf of Aqaba were loosely assigned to the first group. The populations growing at high elevations in South Sinai, under lower temperature and higher humidity, were characterized by a higher number of total and polymorphic ISSR markers compared with other populations. Unique ISSR markers were more often observed in the fingerprinting of seven populations including five populations growing in the high mountains of Saint Catherine in South Sinai and two populations growing at low elevations but at Wadi Hof south-east of Cairo. Interestingly, unique bands were found in the populations that possessed traits associated with larger plant size and seed yield as well as better vigour. These are important criteria for the selection of A. fragrantissima populations for conservation and sustainable commercial use.