The recent emergence of Plasmodium falciparum (Pf) parasites resistant to artemisinin-based combination therapies (ACT) in Africa has outlined the need for continuous molecular surveillance of artemisinin partial resistance. Here, the genetic polymorphism in the Kelch 13 gene (pfk13) and its structural impact were analyzed. PfDNA was extracted from dried blood spots of symptomatic and asymptomatic individuals living in different epidemiological facets of Cameroon. The pfk13 gene was amplified by nested polymerase chain reaction, and amplicons were sequenced to detect single nucleotide polymorphisms (SNPs). The evolutionary history and the impact of the polymorphisms on physicochemical properties, structure, and function of the pfK13 protein were appraised using various in silico models. A total of ten SNPs were identified in this study, of which five non-synonymous SNPs have not been previously reported (L647F, D648V, N657S, K658R, and L663P). The genetic diversity of pfk13 sequences was low, and the pfk13 gene evolved under the neutral model. Some mutations, especially L663P, appeared to affect the function and structure of the pfK13 protein. Analysis of the physicochemical properties of the Cameroonian pfK13 protein sequences revealed slight changes in the solvent-accessible surface area, isoelectric point, and hydrophobicity. The results support the ongoing use of ACTs in the study areas, given the absence of validated SNPs associated with artemisinin partial resistance. Computational findings suggest a possible deleterious effect of some novel SNPs on the pfK13 structure and/or function.