We studied the mitochondrial DNA (mtDNA) polymorphism of 304 Macaca fascicularis fascicularis (M. f. fascicularis) individuals, representative of 4 cynomolgus macaque populations (Indochina, Indonesia, Philippines, and Mauritius). By sequencing a 590-bp fragment in the hypervariable II region of the D-loop region, we defined 70 haplotypes. The homologous region was also characterized in 22 Chinese Macaca mulatta and 2 Macaca sylvanus. The phylogenetic analysis confirms the monophyly of M. f. fascicularis and defines 2 haplotype groups inside the M. f. fascicularis clade: one "insular," encompassing 6 Philippines, 2 Mauritius, and 31 Indonesian haplotypes, the other "continental" that contains all Indochinese and 6 Indonesian haplotypes. Continental and insular group divergence time was estimated to be approximately 10(6) years before present (BP). Among Indonesian haplotypes, some have a continental origin. This suggests either direct migration from mainland to Indonesia or that remnant lineages from an ancient population genetically close to the mainland (i.e., in the Sunda Shelf, <550 000 years BP) were subsequently brought southward to Indonesia. The low nucleotide diversity in the Philippines population suggests a bottleneck following colonization by Indonesian individuals, around 110 000 years BP. mtDNA and further observations of nuclear genetic data corroborate the mixed origin (Indonesian/continental) hypothesis of Mauritius individuals and a population bottleneck.