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Background: The Levant is a crucial region in understanding human migrations between Africa and Eurasia. Although some mitochondrial DNA (mtDNA) studies have been carried out in this region, they have not included the Jordan area. This paper deals with the mtDNA composition of two Jordan populations., Aim: The main objectives of this article are: first, to report mtDNA sequences of an urban and an isolate sample from Jordan and, second, to compare them with each other and with other nearby populations., Subjects and Methods: The analyses are based on HVSI and HVSII mtDNA sequences and diagnostic RFLPs to unequivocally classify into haplogroups 101 Amman and 44 Dead Sea unrelated individuals from Jordan., Results: Statistical analysis revealed that, whereas the sample from Amman did not significantly differ from their Levantine neighbours, the Dead Sea sample clearly behaved as a genetic outlier in the region. Its outstanding Eurasian haplogroup U3 frequency (39%) and its south-Saharan Africa lineages (19%) are the highest in the Middle East. On the contrary, the lack ((preHV)1) or comparatively low frequency (J and T) of Neolithic lineages is also striking. Although strong drift by geographic isolation could explain the anomalous mtDNA pool of the Dead Sea sample, the fact that its mtDNA lineage composition mirrors, in geographic origin and haplogroup frequencies, its Y-chromosome pool, points to founder effect as the main cause. Ancestral M1 lineages detected in Jordan that have affinities with those recently found in Northwest but not East Africa question the African origin of the M1 haplogroup., Conclusion: Results are in agreement with an old human settlement in the Jordan region. However, in spite of the attested migratory spreads, genetically divergent populations, such as that of the Dead Sea, still exist in the area.