has emerged as an important zoonotic parasite that causes persistent symptomatic malaria in humans. The signs and symptoms of malaria are attributed to the blood stages of the parasites, which start from the invasion of erythrocytes by the blood stage merozoites. The apical membrane protein 1 (AMA-1) plays an important role in the invasion. In this study, we constructed and expressed recombinant PkAMA-1 domain II (PkAMA-1-DII) representing the predominant haplotypes from Peninsular Malaysia and Malaysian Borneo and raised specific antibodies against the recombinant proteins in rabbits. Despite the minor amino acid sequence variation, antibodies raised against haplotypes from Peninsular Malaysia and Malaysian Borneo demonstrated different invasion inhibition (46.81% and 39.45%, respectively) to A1-H.1, a reference strain derived from Peninsular Malaysia. Here, we demonstrated how a minor variation in a conserved parasite protein could cast a significant impact on parasite invasion biology, suggesting a complex host-switching of from different locations. This may challenge the implementation of a standardized One Health approach against the transmission of knowlesi malaria.