Genomic surveillance reveals co-occurrence of Plasmodium falciparum drug resistance variants across diverse transmission settings in Ethiopia

The emergence of antimalarial drug resistance threatens malaria control and elimination efforts in Africa. Ethiopia, once a success story in case reduction, is now experiencing a resurgence. Here we examine key drug resistance genes ( Pfmdr1 , Pfcrt , Pfk13 , Pfdhfr and Pfdhps ) and mitochondrial genomes from 605 Plasmodium falciparum isolates collected across 15 districts in Ethiopia with varying transmission intensity and Plasmodium vivax co-endemicity. Although chloroquine was withdrawn for P. falciparum long ago, it remains the first-line treatment for P. vivax ; this overlapping use may shape selection pressure in co-endemic settings that influences resistance markers to artemether–lumefantrine, the current first-line therapy for P. falciparum . A dominant Pf MDR1 NFSND haplotype, associated with reduced lumefantrine susceptibility, was identified alongside near fixation of the chloroquine-resistant Pf CRT CVIET haplotype in specific areas. Concerningly, Pf K13 variants associated with partial artemisinin resistance, R622I (10%), A675V (1.7%) and P441L (1.1%), were expanding. Multilevel models demonstrated robust, independent associations of R622I with Pf CRT CVIET and Pf DHFR AICNI, while ecological predictors were weaker and less consistent. These findings highlight genetic co-occurrence of Pfcrt and Pfk13 mutations in P. vivax – P. falciparum co-endemic settings and can inform antimalarial policy in Ethiopia.