Heteroplasmy, the presence of multiple mitochondrial DNA (mtDNA) haplotypes within cells of an individual, is caused by mutation or paternal leakage. However, heteroplasmy is usually resolved to homoplasmy within a few generations because of germ-line bottlenecks; therefore, instances of heteroplasmy are limited in nature. Here, we report heteroplasmy in the ricefish species Oryzias matanensis, endemic to Lake Matano, an ancient lake in Sulawesi Island, in which one individual was known to have many heterozygous sites in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. In this study, we cloned the ND2 gene for some additional individuals with heterozygous sites and demonstrated that they are truly heteroplasmic. Phylogenetic analysis revealed that the extra haplotype within the heteroplasmic O. matanensis individuals clustered with haplotypes of O. marmoratus, a congeneric species inhabiting adjacent lakes. This indicated that the heteroplasmy originated from paternal leakage due to interspecific hybridisation. The extra haplotype was unique and contained two nonsynonymous substitutions. These findings demonstrate that this hybridisation-driven heteroplasmy was maintained across generations for a long time to the extent that the extra mitochondria evolved within the new host.