Tackling relapsing Plasmodium vivax and zoonotic Plasmodium knowlesi infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches, particularly for P. vivax. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted strain of P. knowlesi and define critical parameters for optimal homology-driven repair. Using iterative rounds of genome editing of the P. knowlesi Duffy binding protein family genes we generate a transgenic line expressing P. vivax Duffy binding protein (PvDBP), a lead vaccine candidate. We demonstrate that PvDBP plays no role in reticulocyte restriction but can alter the macaque/human host cell tropism of P. knowlesi. Critically, antibodies raised against the P. vivax antigen potently inhibit proliferation of this strain, providing an invaluable tool to support vaccine development.