The cultural phenomenon of Crypto-Judaism, defined as the secret adherence to Judaism while publicly professing another faith, arose in Portugal in the beginning of the 16th century after the Decree of Expulsion and the establishment of the Inquisition. Surprisingly, the scientific community acknowledged the persistence of Crypto-Judaic communities at the beginning of the 20th century in central and north-eastern regions of the country (e.g. Bragança). In the present work, we have sampled 56 unrelated individuals from the Bragança Jewish community aiming to characterize their maternal lineage. A 3348bp mtDNA fragment was amplified and sequenced using mitochondrial-specific primers in order to obtain the entire control region. Haplogroup classification was performed according to current nomenclature. High frequencies were found for haplogroups H, HV0, T2, U2 and N1, indicating some degree of European admixture along with a remarkable signature of a Near East ancestry. These data confirm that the Crypto-Jews from Bragança were able to maintain not only their cultural identity but also some ancestral genetic identity, showing a significant population substructure within Portugal, with forensic relevance.