Contemporary Jews comprise an aggregate of ethno-religious communities whose worldwide members identify with each other through various shared religious, historical and cultural traditions 1,2. Historical evidence suggests common origins in the Middle East, followed by migrations leading to the establishment of communities of Jews in Europe, Africa and Asia, in what is termed the Jewish Diaspora3-5. This complex demographic history imposes special challenges in attempting to address the genetic structure of the Jewish people6. Although many genetic studies have shed light on Jewish origins and on diseases prevalent among Jewish communities, including studies focusing on uniparentally and biparentally inherited markers7-16, genome-wide patterns of variation across the vast geographic span of Jewish Diaspora communities and their respective neighbours have yet to be addressed. Here we use high-density bead arrays to genotype individuals from 14 Jewish Diaspora communities and compare these patterns of genome-wide diversity with those from 69 Old World non-Jewish populations, of which 25 have not previously been reported. These samples were carefully chosen to provide comprehensive comparisons between Jewish and non-Jewish populations in the Diaspora, as well as with non-Jewish populations from the Middle East and north Africa. Principal component and structure-like analyses identify previously unrecognized genetic substructure within the Middle East. Most Jewish samples form a remarkably tight subcluster that overlies Druze and Cypriot samples but not samples from other Levantine populations or paired Diaspora host populations. In contrast, Ethiopian Jews (Beta Israel) and Indian Jews (Bene Israel and Cochini) cluster with neighbouring autochthonous populations in Ethiopia and western India, respectively, despite a clear paternal link between the Bene Israel and the Levant. These results cast light on the variegated genetic architecture of the Middle East, and trace the origins of most Jewish Diaspora communities to the Levant.