Background and Objectives Duffy blood group is of major interest in clinical medicine as it is not only involved in blood-transfusion risks and occasionally in neonatal haemolytic disease, but it is also the receptor for the human malaria parasite Plasmodium vivax in the erythrocyte invasion. The aim of this study was to develop a rapid and inexpensive approach for high-throughput Duffy genotyping. Materials and Methods This paper reported the development of a Duffy genotyping assay based on multiplex real-time polymerase chain reaction (PCR) using SYBR Green I fluorescent dye. Results By using this approach for Duffy genotyping we obtained the same results as that for the conventional allele-specific PCR, however, in a high-throughput assay. The Duffy genotyping of field samples demonstrated that P. vivax-infected individuals showed a significantly higher prevalence of two functional alleles than Plasmodium falciparum-infected and non-infected individuals. This finding corroborates the hypothesis that the presence of two functional alleles increases the risk of P. vivax infection. Conclusions This methodology may be suitable for epidemiological studies, particularly for exploring the relationship between Duffy alleles and malaria susceptibility, and also for identification of transfusional incompatibility in blood banks.