Mitochondrial DNA (mtDNA) is maternally transmitted in animals and therefore, individuals are expected to have a single mtDNA haplotype (homoplasmy). Yet, heteroplasmic individuals have been observed in a large number of animal species. Heteroplasmy may emerge as a result of somatic mtDNA mutations, paternal leakage during fertilization or be inherited from a heteroplasmic mother. Understanding the causes of heteroplasmy could shed light into the evolution of mtDNA inheritance. In this study we examined heteroplasmy in progeny from heterospecific crosses of Drosophila for two consecutive generations. We studied the generation of heteroplasmy from paternal leakage and the maternal transmission of heteroplasmy. Our data reveal non-random patterns in the emergence and transmission of heteroplasmy and suggest that heteroplasmy depends on the family of origin.