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The expression of the Duffy Antigen/Receptor for Chemokines (DARC) on red blood cells (RBC) has been commonly determined using hemagglutination tests. Because the vast majority of African individuals are Duffy-negative, screening for DARC expression on RBC is a valuable tool to assess Caucasian admixture in populations of African descent. Furthermore, blood group antigens have been frequently tested as potential risk factors for complex diseases. We established a dot-blotting protocol using sequence-specific oligonucleotides (SSOs) for the DARC-46T (“Duffy-positive”) and -46C (“Duffy-negative”) alleles. With this method, but not with serological methods, Duffy-positive individuals can be further characterized as homozygous or heterozygous for the dominant Duffy-positive allele, allowing more precise estimation of allele frequencies and admixture in heterogeneous populations. In unrelated African American ( n = 235), Afro-Caribbean ( n = 90) and Colombian ( n = 93) subjects, the frequency of the -46T allele was 21.7%, 12.2% and 74.7%, respectively. The percentage of Duffy-positive individuals (homozygous or heterozygous for the −46T allele) in each population was in accordance with published frequencies. As expected, the -46C allele was not detected in 20 Caucasian subjects. This sensitive and specific method allows for the rapid and inexpensive screening of large samples for Duffy genotypes using small quantities of genomic DNA.