Jordan, located in the Levant region, is an area crucial for the investigation of human migration between Africa and Eurasia. However, the genetic history of Jordanians has yet to be clarified, including the origin of the Bedouins today resident in Jordan. Here, we provide new genetic data on autosomal independent markers in two Jordanian population samples (Bedouins and the general population) to begin to examine the genetic diversity inside this country and to provide new information about the genetic position of these populations in the context of the Mediterranean and Middle East area. The markers analyzed were 18 Alu polymorphic insertions characterized by their identity by descent, known ancestral state (lack of insertion), and apparent selective neutrality. The results indicate significant genetic differences between Bedouins and general Jordanians (p = 0.038). Whereas Bedouins show a close genetic proximity to North Africans, general Jordanians appear genetically more similar to other Middle East populations. In general, these data are consistent with the hypothesis that Bedouins had an important role in the peopling of Jordan and constitute the original substrate of the current population. However, migration into Jordan in recent years likely has contributed to the diversity among current Jordanian population groups.