Several unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na+ pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous pkatp4 locus was replaced by a recodonized P. knowlesi atp4 (pkatp4) coding region or the orthologous coding region from P. falciparum, Plasmodium malariae, Plasmodium ovale subsp. curtisi, or Plasmodium vivax. Each OR transgenic line displayed a similar growth pattern to the parental P. knowlesi line. We found significant orthologue-specific differences in parasite susceptibility to three chemically unrelated ATP4 inhibitors, but not to comparator drugs, among the P. knowlesi OR lines. The PfATP4OR transgenic line of P. knowlesi was significantly more susceptible than our control PkATP4OR line to three ATP4 inhibitors: cipargamin, PA21A092, and SJ733. The PvATP4OR and PmATP4OR lines were similarly susceptible to the control PkATP4OR line, but the PocATP4OR line was significantly less susceptible to all ATP4 inhibitors than the PkATP4OR line. Cipargamin-induced inhibition of Na+ efflux was also significantly greater with the P. falciparum orthologue of ATP4. This confirms that species-specific susceptibility differences previously observed in ex vivo studies of human isolates are partly or wholly enshrined in the primary amino acid sequences of the respective ATP4 orthologues and highlights the need to monitor efficacy of investigational malaria drugs against multiple species. P. knowlesi is now established as an important in vitro model for studying drug susceptibility in non-falciparum malaria parasites. IMPORTANCE Effective drugs are vital to minimize the illness and death caused by malaria. Development of new drugs becomes ever more urgent as drug resistance emerges. Among promising compounds now being developed to treat malaria are several unrelated molecules that each inhibit the same protein in the malaria parasite-ATP4. Here, we exploited the genetic tractability of P. knowlesi to replace its own ATP4 genes with orthologues from five human-infective species to understand the drug susceptibility differences among these parasites. We previously estimated the susceptibility to ATP4-targeting drugs of each species using clinical samples from malaria patients. These estimates closely matched those of the corresponding "hybrid" P. knowlesi parasites carrying introduced ATP4 genes. Thus, species-specific ATP4 inhibitor efficacy is directly determined by the sequence of the gene. Our novel approach to understanding cross-species susceptibility/resistance can strongly support the effort to develop antimalarials that effectively target all human malaria parasite species.