Inter-specific hybrid zones for Hylobates gibbons are known in Southeast Asia. Among these, one hybrid zone between Hylobates lar and H. pileatus is located in Khao Yai National Park, Thailand. To find molecular evidence for the natural hybridization of the gibbons in this region, we studied mitochondrial DNA (mtDNA) of 68 gibbons of the H. lar phenotype living adjacent to the hybrid zone. Nucleotide sequencing of a fragment of mtDNA spanning hyper variable segment I showed that nine gibbons had an mtDNA haplotype of H. pileatus, and that seven of these nine gibbons belonged to a single maternal lineage over three generations. It is thus confirmed that introgression between H. lar and H. pileatus exists and the initial hybridization took place ages ago.