A molecular marker of artemisinin-resistant Plasmodium falciparum malaria Artemisinin resistance in Plasmodium falciparum in Southeast Asia threatens global malaria control. A molecular marker is needed to monitor the spread of resistance. Using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical isolates from Cambodia, mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') were associated with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant allele correlates with the spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates, and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. The emergence of artemisinin-resistant Plasmodium falciparum in Cambodia threatens global malaria control. The risk of resistance spreading from western Cambodia to the Greater Mekong Subregion and Africa is extremely worrisome. Clinical resistance is defined by reduced parasite clearance rates or persistence of microscopically detectable parasites on the third day of treatment. The half-life parameter correlates with in vitro and ex vivo survival rates. However, the lack of a molecular marker hampers containment and detection of resistant parasites. Recent genome-wide analyses suggest recent positive selection in areas of resistance. No reliable molecular marker has been identified. One possible explanation is that parasite clearance half-life is influenced by developmental stage and host factors. Genome-wide association studies are confounded by uncertainties about population structure. Recent evidence suggests distinct emergence events. An alternative strategy is to analyze mutations in laboratory-adapted parasite clones and use this information to guide analysis of clinical isolates. This strategy was used to explore molecular signatures of clinical resistance in Cambodia. A candidate molecular marker of artemisinin resistance was identified. The ART-resistant F32-ART5 parasite line was selected by culturing the ART-sensitive F32-Tanzania clone under a dose-escalating regimen of artemisinin. Whole-genome sequences were obtained for both F32-ART5 and F32-TEM. Mutations in the PF3D7_1343700 gene were identified. These mutations were associated with increased survival rates. Analysis of sequence polymorphism in seven genes showed that PF3D7_1343700 polymorphism is significantly associated with in vitro survival rates. The K13-propeller domain shows a significant association with survival rates. The frequency of mutant alleles increasedA molecular marker of artemisinin-resistant Plasmodium falciparum malaria Artemisinin resistance in Plasmodium falciparum in Southeast Asia threatens global malaria control. A molecular marker is needed to monitor the spread of resistance. Using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical isolates from Cambodia, mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') were associated with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant allele correlates with the spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates, and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. The emergence of artemisinin-resistant Plasmodium falciparum in Cambodia threatens global malaria control. The risk of resistance spreading from western Cambodia to the Greater Mekong Subregion and Africa is extremely worrisome. Clinical resistance is defined by reduced parasite clearance rates or persistence of microscopically detectable parasites on the third day of treatment. The half-life parameter correlates with in vitro and ex vivo survival rates. However, the lack of a molecular marker hampers containment and detection of resistant parasites. Recent genome-wide analyses suggest recent positive selection in areas of resistance. No reliable molecular marker has been identified. One possible explanation is that parasite clearance half-life is influenced by developmental stage and host factors. Genome-wide association studies are confounded by uncertainties about population structure. Recent evidence suggests distinct emergence events. An alternative strategy is to analyze mutations in laboratory-adapted parasite clones and use this information to guide analysis of clinical isolates. This strategy was used to explore molecular signatures of clinical resistance in Cambodia. A candidate molecular marker of artemisinin resistance was identified. The ART-resistant F32-ART5 parasite line was selected by culturing the ART-sensitive F32-Tanzania clone under a dose-escalating regimen of artemisinin. Whole-genome sequences were obtained for both F32-ART5 and F32-TEM. Mutations in the PF3D7_1343700 gene were identified. These mutations were associated with increased survival rates. Analysis of sequence polymorphism in seven genes showed that PF3D7_1343700 polymorphism is significantly associated with in vitro survival rates. The K13-propeller domain shows a significant association with survival rates. The frequency of mutant alleles increased