Malaria is a parasitic disease caused predominantly by two species in humans: Plasmodium falciparum and P. vivax. These parasites have a complicated multi-host lifecycle, in humans this includes a continuous asexual replication within the blood which results in clinical disease. This begins when the invasive form of parasite, called a merozoite, attaches to and infects an erythrocyte in which they replicate to form the daughter parasites. There is still much to understand regarding merozoite cell-entry biology, particularly for P. vivax which is yet to be adapted to in vitro culture. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to erythrocytes and have long been considered as potential vaccine targets. However, the function of most MSPs has yet to be defined. Here, I applied targeted gene editing to investigate MSP4 and 5 function in the in vitro culturable P. falciparum and P. knowlesi, a close relative of P. vivax. These proteins likely arose from a gene duplication event as their chromosomal locus is highly conserved and there is structural similarity, hence they may have a linked or complementary biological role. CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP5 is refractory to gene deletion, but it could be functionally replaced by P. vivax MSP5. Proceeding to conditional knock-down of MSP5 protein expression revealed a severe cell-entry defect. Conversely, deletion of P. knowlesi MSP4 had no phenotype. We, and others, found the opposite for P. falciparum where MSP4 is essential but MSP5 is dispensable. This study provides a range of gene-edited lines to investigate MSP4 and MSP5 function and immunological importance and emphasizes that vaccine candidates must be considered individually for the two most prominent human malarias, while promoting MSP5 as a potential vaccine candidate for P. vivax.