Measures to combat the parasites that cause malaria have become compromised because of reliance on a small arsenal of drugs and emerging drug resistance. We conducted a transposon mutagenesis screen in the primate malaria parasite Plasmodium knowlesi, producing the most complete classification of gene essentiality in any Plasmodium spp. to date, with the resolution to define truncatable genes. We found conservation in the druggable genome between Plasmodium spp. and divergences in mitochondrial metabolism. Perturbation analyses with the frontline antimalarial artemisinin revealed modulators that both increase and decrease drug susceptibility. Our findings aid prioritization of drug and vaccine targets for the Plasmodium vivax clade and reveal mechanisms of resistance that can inform therapeutic development.