Heteroplasmy, the coexistence of two types of mitochondria, is sometimes observed in hybrid embryos due to interspecific crosses. However, heteroplasmy is usually rapidly removed from populations, because it reduces individual fitness due to “mitonuclear incompatibility”. In this study, we report that heteroplasmy may have been maintained across generations in Oryzias matanensis, a medaka species endemic to Lake Matano, Sulawesi Island. We found by cloning of ND2 gene that about 35% of individuals are heteroplasmic. De novo assembling of short read sequences successfully reconstructed two mitochondrial genomes in heteroplasmic individuals. Phylogenetic analyses showed that two mitochondrial haplotypes were distant from each other, and that one of them clustered with those of O. marmoratus, a related species inhabiting neighbor lakes, suggesting that the heteroplasmy in O. matanensis originated from ancient hybridization between these two species. We also found that no stop codon was present in all protein coding genes of both mitochondrial genomes, suggesting that the two mitochondria function within heteroplasmic individuals. Interestingly, the mitochondria originating from O. marmoratus had uniquely evolved within the new host, O. matanensis, and many of the substitutions were non-synonymous. We therefore hypothesized that introgressed mitochondria have acquired new functions to avoid mitonuclear incompatibility.