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The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases, and measured ring-stage survival rates following drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13–49% to 0.3–2.4% following the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2–29% following the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared to reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.