This study was funded fully or partially with funds from FAPESPA/VALE (Funda??o Amaz?nia de Amparo a Estudos e Pesquisas and VALE) and UFPA (Universidade Federal do Par?) under grant number ICAAF 070/2010 and FAPESPA/PPSUS ICAAF 182/2012 coordinated by MGC; and CAPES (Coordenac?o de Aperfei?oamento de Pessoal de N?vel Superior) and UFPA?Rede de Pesquisa em Genomica Populacional Humana (RPGPH)-051/2013 coordinated by ARS. ARS is supported by CNPq (Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico/CNPq/Productivity 304413/2015-1) Universidade Federal do Par?. Laborat?rio de Gen?tica Humana e M?dica. Bel?m, PA, Brazil / Funda??o Centro de Hemoterapia e Hematologia do Par?. Bel?m, PA, Brazil. Universidade de Bras?lia, Bras?lia. Distrito Federal, DF, Brazil. Universidade Federal do Par?. Laborat?rio de Gen?tica Humana e M?dica. Bel?m, PA, Brazil. Universidade Federal do Par?. Laborat?rio de Gen?tica Humana e M?dica. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal do Par?. Laborat?rio de Microbiologia e Imunologia. Bel?m, PA, Brazil. Universidade Federal do Par?. Laborat?rio de Gen?tica Humana e M?dica. Bel?m, PA, Brazil. Background: Malaria can be transmitted by blood transfusion through donations collected from asymptomatic or parasitic donors. The parasites are released into the bloodstream during its life cycle and will therefore be present in donated blood by infected individuals. All cases of transfusion-transmitted malaria (TTM) notifed since 2005 in Brazil were fatal. A good screening tool for Plasmodium spp. detection in blood units must have a high detection threshold, and the prevention of TTM relies entirely on the exclusion of potentially infected donors. However, in Brazilian blood banks, the screening test relies on blood thick smears examination. Methods: The molecular diagnostic based on mitochondrial DNA (mtDNA) using real time PCR (mt-qPCR) was improved to detect Plasmodium falciparum, Plasmodium vivax, and standardized for use in Plasmodium malariae. The analytic sensitivity of this mt-qPCR methodology was performed using a sample of P. vivax. Results: The mt-qPCR was highly efcient, and the analytic sensitivity for P. vivax was determined (0.000006 para? sites/?L). This method was tested to detect P. vivax and P. falciparum in individuals from two malaria-endemic areas in Brazil, Amazon region (Par? and Rond?nia states), the samples were collected in 10 reference units of two blood banks (Par?/nine cities and Rond?nia/Porto Velho), and parasites mtDNA were detected in 10 of 2224 potential blood donors (0.45%). In all 10 positive samples, only P. vivax was detected. Conclusion: Molecular diagnostic using mt-qPCR was efective in revealing infected potential donors with good per? spectives to be applied as screening routine of asymptomatic carriers for preventing transfusion-transmitted malaria in blood banks.