BackgroundPfcrtgene has been associated with chloroquine resistance and thepfmdr1gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether–lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria,pfcrthaplotype, andpfmdr1single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission.Methods230 microscopically confirmedP. falciparumisolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence ofpfcrthaplotypes andpfmdr1SNPs. Furthermore, thepfmdr1gene copy number (CNV) was determined using real-time PCR. AP-value of less or equal to 0.05 was considered significant.ResultsOf the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM forpfcrthaplotype,pfmdr1-86,pfmdr1-184,pfmdr1-1042 andpfmdr1-1246, respectively. The mutantpfcrthaplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively.Plasmodium falciparumwith chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4,P= 0.00).Pfmdr1-N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at thepfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eightpfmdr1haplotypes at codons N86Y–Y184F–D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution ofpfmdr1SNPs, haplotypes, and CNV between the two study sites (P> 0.05).ConclusionPlasmodium falciparumwith thepfcrtwild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y–Y184F–D1246Y. A continuous investigation is needed to closely monitor the changes in thepfmdr1SNPs, which are associated with the selection of parasite populations by ACT.