One of the key obstacles to malaria elimination is largely attributed toPlasmodium vivax’sability to form resilient hypnozoites in the host liver that cause relapsing infections. As a result, interruption ofP. vivaxtransmission is difficult.P. vivaxtransmission occurs in Duffy-positive individuals and have been mainly thought to be absent in Africa. However, increasing studies using molecular tools detectedP. vivaxamong Duffy-negative individuals in various African countries. Studies on the AfricanP. vivaxhas been severely limited because most of malaria control program focus mainly on falciparum malaria. In addition, there is a scarcity of laboratory infrastructures to overcome the biological obstacles posed byP. vivax. Herein, we established field transmission of EthiopianP. vivaxfor routine sporozoite supply followed by liver stage infection in Mali. Furthermore, we evaluated localP. vivaxhypnozoites and schizonts susceptibilities to reference antimalarial drugs. The study enabled the assessment of local AfricanP. vivaxhypnozoite production dynamics. Our data displayed the ability of the AfricanP. vivaxto produce hypnozoite formsex-vivoat different rates per field isolate. We report that while tafenoquine (1µM) potently inhibited both hypnozoites and schizont forms; atovaquone (0.25µM) and the phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691 (0.5µM) showed no activity against hypnozoites forms. Unlike hypnozoites forms,P. vivaxschizont stages were fully susceptible to both atovaquone (0.25µM) and the (PI4K)-specific inhibitor KDU691 (0.5µM). Together, the data revealed the importance of the local platform for further biological investigation and implementation of drug discovery program on the AfricanP. vivaxclinical isolates.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12879-023-08381-y.