Duffy blood group system is of interest in several fields of science including transfusion medicine, immunology and malariology. Although some methods have been developed for Duffy polymorphism genotyping, not all of them have been sufficiently described and validated, and all present limitations. At the same time, the frequency of Duffy alleles and antigens in some densely populated regions of the world are still missing. In this study we present new tests for genotyping the major alleles of the Duffy blood system and describe Duffy alleles and antigens in blood donors and transfusion-dependent patients in Minas Gerais, Brazil. A simple and reproducible strategy was devised for Duffy genotyping based on real-time PCR that included SNPs rs12075 and rs2814778. No significant differences between the allele frequencies were observed comparing blood donors and patients. Among the blood donors, the phenotype Fy(a-b+) was the most common and the Fy(a-b-) phenotype, associated with populations of African descent, was remarkably less common among subjects who self-identified as black in comparison to other ethnoracial categories. However, the African ancestry estimated by molecular markers was significantly higher in individuals with the allele associated to the Duffy null phenotype. The genotyping method presented may be useful to study Duffy genotypes accurately in different contexts and populations. The results suggest a reduced risk of alloimmunization for Duffy antigens and increased susceptibility for malaria in Minas Gerais, considering the high frequency of Duffy-positive individuals.