Mutations in the () gene are linked to delayed parasite clearance in response to artemisinin-based combination therapies (ACTs) in Southeast Asia. To explore the evolutionary rate and constraints acting on this gene, orthologs from species sharing a recent common ancestor with and were analyzed. These comparative studies were followed by genetic polymorphism analyses within using 982 complete sequences from public databases and new data obtained by next-generation sequencing from African and Haitian isolates. Although orthologs evolve at heterogeneous rates, the gene was conserved across the genus, with only synonymous substitutions being found at residues where mutations linked to the delayed parasite clearance phenotype have been reported. This suggests that those residues were under constraint from undergoing nonsynonymous changes during evolution of the genus. No fixed nonsynonymous differences were found between and its orthologs in closely related species found in African apes. This indicates that all nonsynonymous substitutions currently found in are younger than the time of divergence between and its closely related species. At the population level, no mutations linked to delayed parasite clearance were found in our samples from Africa and Haiti. However, there is a high number of single mutations segregating in populations, and two predominant alleles are distributed worldwide. This pattern is discussed in terms of how changes in the efficacy of natural selection, affected by population expansion, may have allowed for the emergence of mutations tolerant to ACTs.