Malaria is still an important challenge for global public health because of its extensive mortality and morbidity. Plasmodium ovale is mainly distributed in tropical regions of Africa and Asia. it includes two distinct ovale malaria species, which are P. ovale curtisi and P. ovale wallikeri. Apical membrane antigen-1 (AMA-1) is an asexual blood-stage protein which is essential for Plasmodium. Thus far, no study on gene polymorphism and immunogenicity of P. ovale AMA-1 (PoAMA-1) has been conducted. Amplified poama1 gene products from 14 P. ovale curtisi samples and 12 P. ovale wallikeri samples imported from Africa to Jiangsu Province, China were sequenced and their polymorphisms were analyzed. We expressed recombinant PoAMA-1 (rPoAMA-1, 53 kDa) proteins in an Escherichia coli expression system and evaluated immune responses against the rPoAMA-1 in BALB/c mice. We identified a synonymous mutation in nucleotide position 333 of the pocama-1 gene and powama-1 did not reveal any variation. The humoral and cellular immune responses to rPoAMA-1 were evaluated using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. rPoAMA-1-immunized mice produced specific antibodies as verified by immunoblotting. The rPoAMA-1 induced high antibody titers (1: 640,000), and had high avidity indexes (an average of 78.63% and 83.40%). The antibodies also recognized the native proteins, namely, crude antigen from blood stages. Cross-reactivity between rPocAMA-1 and rPowAMA-1 was observed. Moreover, rPoAMA-1s induced interferon (IFN)-gamma-secreting cells in mice and increased lymphocyte proliferation response. Low genetic diversity was observed in poama-1 from the Jiangsu Province imported malaria cases, and further studies conclusively showed its strong immunogenicity. Significant cross-reactivity was found between rPocAMA-1 and rPowAMA-1, suggesting that a single PoAMA-1 antigen could be used to diagnose P. ovale curtisi or P. ovale wallikeri patient simultaneously. However, further evaluation needs to be carried out to validate the potential and limitations of PoAMA-1 as a candidate vaccine.