The establishment of Jewish communities in the territory of contemporary Portugal is archaeologically documented since the 3rd century CE, but their settlement in Trás-os-Montes (NE Portugal) has not been proved before the 12th century. The Decree of Expulsion followed by the establishment of the Inquisition, both around the beginning of the 16th century, accounted for a significant exodus, as well as the establishment of crypto-Jewish communities. Previous Y chromosome studies have shown that different Jewish communities share a common origin in the Near East, although they can be quite heterogeneous as a consequence of genetic drift and different levels of admixture with their respective host populations. To characterize the genetic composition of the Portuguese Jewish communities from Trás-os-Montes, we have examined 57 unrelated Jewish males, with a high-resolution Y-chromosome typing strategy, comprising 16 STRs and 23 SNPs. A high lineage diversity was found, at both haplotype and haplogroup levels (98.74 and 82.83%, respectively), demonstrating the absence of either strong drift or founder effects. A deeper and more detailed investigation is required to clarify how these communities avoided the expected inbreeding caused by over four centuries of religious repression. Concerning haplogroup lineages, we detected some admixture with the Western European non-Jewish populations (R1b1b2-M269, ∼28%), along with a strong ancestral component reflecting their origin in the Middle East [J1(xJ1a-M267), ∼12%; J2-M172, ∼25%; T-M70, ∼16%] and in consequence Trás-os-Montes Jews were found to be more closely related with other Jewish groups, rather than with the Portuguese non-Jewish population. Am J Phys Anthropol 2010. © 2009 Wiley-Liss, Inc.