Ethiopia is central to population genetic studies investigating the out of Africa expansion of modern humans, as shown by Y chromosome and mtDNA studies. To address the level of genetic differentiation within Ethiopia, and its relationship to Sub-Saharan Africa and Eurasia, we studied an 8 kb segment of the X-chromosome from 72 chromosomes from the Amhara, Oromo and Ethiopian Jews, and compared these results with 804 chromosomes from Middle Eastern, African, Asian and European populations, and 22 newly typed Saharawi. Within Ethiopia the two largest ethnic groups, the Amhara and Oromo, were not found to be statistically distinct, based on an exact test of haplotype frequencies. The Ethiopian Jews appear as an admixed population, possibly of Jewish origin, though the data remain equivocal. There is evidence of a close relationship between Ethiopian and Yemenite Jews, likely a result of indirect gene flow. Within an African and Eurasian context, the distribution of alleles of a variable T(n) repeat, and the spread of haplotypes containing Africa-specific alleles, provide evidence of a genetic continuity from Sub-Saharan Africa to the Near East, and furthermore suggest that a bottleneck occurred in Ethiopia associated with an out of Africa expansion. Ethiopian genetic heterogeneity, as evidenced by principal component analysis of haplotype frequencies, most likely resulted from periods of subsequent admixture. While these results are from the analysis of one locus, we feel that in association with data from other marker systems they add a complementary perspective on the history of Ethiopia.