Heteroplasmy, the presence of multiple mitochondrial DNA 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. In Oryzias matanensis, a medaka species endemic to Lake Matano, an ancient lake in Sulawesi Island, some individuals are known to be heteroplasmic, and it has been suggested that this heteroplasmy originated from ancient hybridization with O. marmoratus, a congeneric species inhabiting adjacent lakes. In this study, we compared the whole mitochondrial and nuclear genome sequences between heteroplasmic and homoplasmic O. matanensis individuals. Molecular evolution analyses of the whole mitochondrial genome demonstrated that the extra mitochondria originating from O. marmoratus have uniquely evolved within the new host under strong positive selection. Population genetic structure analyses based on genome-wide single nucleotide polymorphisms revealed that homo- and heteroplasmic individuals are clearly separated from each other, suggesting the presence of reproductive isolation between them. Genome-wide scan of F_ST detected some regions completely segregated between homo- and heteroplasmic individuals (i.e., F_ST = 1). These findings suggest that heteroplasmy caused by ancient hybridization may have led to mitonuclear coevolution and triggered speciation.