It has been discovered that Plasmodium knowlesi (P. knowlesi) is transmitted from macaque to man. Thus, the aim of the present study was to determine P. knowlesi genetic diversity in both human (n = 147) and long-tailed macaque (n = 26) samples from high- and low-endemicity localities. Genotyping was performed using seven neutral microsatellite loci markers. The size of the alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (HE), linkage disequilibrium (LD), and genetic differentiation (FST) were determined. In highly endemic P. knowlesi localities, the MOI for human and long-tailed macaque isolates was 1.04 and 1.15, respectively, while the Na was 11.14 and 7.86, respectively. Based on the allele frequency distribution for all loci, and with FST