ObjectivesPlasmodium vivax malaria was thought to be rare in Africans who lack the Duffy blood group antigen expression. However, recent studies indicate that P. vivax can infect Duffy-negative individuals and has spread into areas of high Duffy negativity across Africa. Our study compared epidemiological and genetic features of P. vivax between African regions. MethodsA standardized approach was used to identify and quantify P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. The study involved sequencing the Duffy binding protein (DBP) gene and inferring genetic relationships among P. vivax populations across Africa. ResultsAmong 1215 febrile patients, the proportions of Duffy negativity ranged from 20–36% in East Africa to 84% in southern Africa. Average P. vivax prevalence among Duffy-negative populations ranged from 9.2% in Sudan to 86% in Botswana. Parasite density in Duffy-negative infections was significantly lower than in Duffy-positive infections. P. vivax in Duffy-negative populations were not monophyletic, with P. vivax in Duffy-negative and Duffy-positive populations sharing similar DBP haplotypes and occurring in multiple, well-supported clades. ConclusionsDuffy-negative Africans are not resistant to P. vivax, and the public health significance of this should not be neglected. Our study highlights the need for a standardized approach and more resources/training directed towards the diagnosis of vivax malaria in Africa.