Plasmodium falciparum resistance to atovaquone-proguanil has so far been associated with Y268S or Y268N mutations in cytochrome b, although these changes were identified in only seven of the 11 treatment failures. Here, we describe 10 new cases of atovaquone-proguanil treatment failures among which the parasite resistance was confirmed in six cases, either by identifying correct plasma drug concentrations or by observing in vitro atovaquone resistance. Resistance was consistently associated with codon 268 mutations (Y268S or a previously unidentified mutation, Y268C). Notably, mutations were not detected before the treatment but only after the drug exposure.