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A major threat to the goal of eliminating malaria, particularly in Southeast Asia, is the spread of Plasmodium falciparum resistant to artemisinin-based combination therapies. P218 is a drug candidate designed to combat antifolate-sensitive and -resistant parasites. However, there is no evidence that P218 is effective against artemisinin-resistant P. falciparum. This report investigated the susceptibilities of 10 parasite isolates from Southeast Asia to P218 and other antimalarial drugs. All isolates with different levels of artemisinin resistance were genetically distinct from one another, although common haplotypes associated with antimalarial resistance were identified. All isolates were highly resistant to pyrimethamine, and none of them were significantly less sensitive to P218 than the pyrimethamine-resistant laboratory strain V1/S. Significant differences in sensitivity to other types of antimalarials (mefloquine, atovaquone and chloroquine) compared with V1/S were found for some isolates, although the differences were not clinically relevant. P218 is thus efficacious against multi-drug (including artemisinin-resistant P. falciparum., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)