The genetic diversity ofpkmsp-1of MalaysianPlasmodium knowlesiisolates was studied recently. However, the study only included three relatively older strains from Peninsular Malaysia and focused mainly on the conserved blocks of this gene. In this study, the full-lengthpkmsp-1sequence of recentP. knowlesiisolates from Peninsular Malaysia was characterized, along with Malaysian Borneo and Thailandpkmsp-1sequences that were retrieved from GenBank. Genomic DNA ofP. knowlesiwas extracted from human blood specimens and thepkmsp-1gene was PCR-amplified, cloned, and sequenced. The sequences were analysed for genetic diversity, departure from neutrality, and geographical clustering. Thepkmsp-1gene was found to be under purifying/negative selection and grouped into three clusters via a neighbour-joining tree and neighbour net inferences. Of the four polymorphic blocks inpkmsp-1, block IV, was most polymorphic, with the highest insertion–deletion (indel) sites. Two allelic families were identified in block IV, thereby highlighting the importance of this block as a promising genotyping marker for the multiplicity of infection study ofP. knowlesimalaria. A single locus marker may provide an alternate, simpler method to typeP. knowlesiin a population.