Objective To analyze the polymorphism of the K13 protein-encoding gene of Plasmodium ovale curtisi and wallikeri isolates from Africa so as to provide molecular basis for monitoring artemisinin resistance of P. ovale in Africa. Methods The blood samples were collected from imported malaria patients with P. ovale identified by microscopic and epidemiological investigation from 2012 to 2016 in Jiangsu Province. DNA was extracted from the blood samples and the P. ovale K13 gene was amplified by nested PCR. The subspecies of P. ovale were further determined by Real-time TaqMan^R PCR. The acquired DNA sequences were aligned with reference sequences from P. ovale curtisi subspecies (PlasmoDB: PocGH01_12019400) and the wallikeri subspecies (GenBank: LT594516.1) using BioEdit software. The mutations of the obtained DNA sequences were analyzed using DNAstar software. The polymorphism of K13 gene was analyzed by DnaSP software. The phylogenetic tree was constructed using MEGA software. Results A total of 168 P. ovale infected blood samples were collected from malaria patients during 2012-2016 in Jiangsu Province. It was confirmed that the sources of infection were from Central African (95 cases), South Africa (37 cases), West Africa (34 cases) and one case from East and North Africa. A 1 500 bp fragment of K13 gene was successfully amplified from all samples and sequenced. The sequence results identified that half of the samples were P. ovale curtisi subspecies (84) or wallikeri subspecies (84). The nucleotide diversity index π of P. ovale K13 gene was 0.000 02. The haplotype diversity index Hd was 0.024. The K13 genes of two subspecies contained two haplotypes and one single nucleotide polymorphism site. There is only one nucleotide polymorphism within each P. ovale subspecies. The P. ovale curtisi had a A/G polymorphism at nucleotide 717 (amino acid 239E) and the P. ovale wallikeri had a T/A polymorphism at the nucleotide 1998 (amino acid 666P), both of them were synonymous mutations without the change of encoded amino acids. The two mutations identified in this study are not related to P. ovale artemisinin resistance. Neutral test was performed on the DNA sequences of both subspecies of P. ovale without statistical difference. The mutation in P. ovale K13 gene in this study is in accordance with the neutral evolution model. The phylogenetic tree shows that the two haplotypes of P. ovale curtisi subspecies are clustered into one branch, and the two haplotypes of P. ovale wallikeri subspecies are clustered into another branch. Conclusion No nonsynonymous mutation is found in K13 gene of P. ovale curtisi and wallikeri subspecies in this study. K13 gene has a low level of genetic polymorphism in Africa isolations and no mutation is related to P. ovale artemisinin resistance.