Genetic data corresponding to four negrito populations (two Aeta and two Agta; n = 120) from the Luzon region of the Philippines have been analyzed. These data comprise mitochondrial DNA (mtDNA) hypervariable segment 1 haplotypes and haplogroups, Y-chromosome haplogroups and short tandem repeats (STRs), autosomal STRs, and X-chromosome STRs. The genetic diversity and structure of the populations were investigated at a local, regional, and interregional level. We found a high level of autosomal differentiation, combined with no significant reduction in diversity, consistent with long-term settlement of the Luzon region by the ancestors of the Agta and Aeta followed by reduced gene flow between these two ethnolinguistic groups. Collectively, the Aeta have a much higher ratio of female:male effective population size than do the Agta, a finding that supports phylogenetic analysis of their mtDNA and Y-chromosome haplogroups, which suggests different genetic sex-biased contributions from putative Austronesian source populations. We propose that factors of social organization that led to the reduction in Agta female effective population size may also be linked to the limited incorporation of female lineages associated with the settlement of the Philippines by Austronesian speakers; conversely, the reduction in Aeta male effective population size, relative to females, could be indicative of a limited incorporation of male lineages associated with this demographic process.