We evaluated the incidence of anti-Dengue virus (DENV) antibodies and dengue viremia in a region of Mexico with a high prevalence of dengue. DENV is the most important arthropod-borne virus in terms of human morbidity and mortality in America We tested 800 blood donors from a tertiary care teaching hospital that provides care in Northeast Mexico, to identify anti-DENV IgM and IgG antibodies by enzyme-linked immunosorbent assay (ELISA) and DENV genome by reverse transcription polymerase chain reaction (RT-PCR). In addition, routine tests for donors including Brucella, Hepatitis C virus (HCV), Venereal Disease Research Laboratory (VDRL), HIV-1 and HBsAg identification were performed. We found that 59% of donors were reactive for anti-DENV IgG and none of them had reported recent DENV infection; however, 16 (2%) were reactive for anti-DENV IgM antibodies. None of them were viremic at the time of donation. Routine tests showed that the prevalence of anti-Brucella was 0.71%, anti-HCV 0.71%, anti-HIV-1-2 0.14%, HBsAg 0.14% and VDRL test 0.57%. Although DENV transmission by blood transfusion had not been confirmed in Mexico, the finding of a high prevalence of anti-DENV IgM-positive donors with asymptomatic manifestations and the recent viremia reported in blood donors suggests that this route of transmission might be possible.