Drug resistance of malaria parasites remains a problem affecting antimalarial treatment and control of the disease. We previously synthesized an antimalarial endoperoxide, N-89, having high antimalarial effects in vitro and in vivo. In this study we seek to understand the resistant mechanism against N-89 by establishing a highly N-89-resistant clone, named NRC10H, of the Plasmodium falciparum FCR-3 strain. We describe gene mutations in the parent FCR-3 strain and the NRC10H clone using whole-genome sequencing and subsequently by expression profiling using quantitative real-time PCR. Seven genes related to drug resistance, proteolysis, glycophosphatidylinositol anchor biosynthesis, and phosphatidylethanolamine biosynthesis exhibited a single amino acid substitution in the NRC10H clone. Among these seven genes, the multidrug resistance protein 2 (mdr2) variant A532S was found only in NRC10H. The genetic status of the P. falciparum endoplasmic reticulum-resident calcium binding protein (PfERC), a potential target of N-89, was similar between the NRC10H clone and the parent FCR-3 strain. These findings suggest that the genetic alterations of the identified seven genes, in particular mdr2, in NRC10H could give rise to resistance of the antimalarial endoperoxide N-89.