Recent adaptation of the zoonotic pathogen Plasmodium knowlesi to continuous culture in human red blood cells (RBCs) provides an opportunity to compare molecular events required for RBC invasion between P. knowlesi and the major human malaria pathogen P. falciparum. In order to facilitate this comparison, we optimised a robust method to isolate viable and invasive P. knowlesi merozoites to high purity and yield. We found that P. knowlesi merozoites have a markedly increased longevity over those from P. falciparum. The larger size of the P. knowlesi merozoite is an advantage in probing cell structure using immunofluorescence, super resolution and electron microscopy imaging. Quantitative invasion inhibition assays demonstrated that most, but not all, invasion inhibitory reagents were inhibitory to both P. knowlesi and P. falciparum merozoites; providing insights into species specific activity. Finally, we identify a class of poly-sulfonate polymer that is able to efficiently inhibit invasion in both species, providing a foundation for potential pan-Plasmodium therapeutic development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second major cause of human malaria-related morbidity, this study paves the way for inter-specific dissection of all three major pathogenic malaria species.