Animal mitochondrial DNA (mtDNA) is predominantly inherited maternally. Various mechanisms to avoid the transmission of paternal mtDNA to offspring have been proposed, including the dilution of paternal mtDNA by maternal mtDNA in the zygote. The effectiveness of dilution as a barrier will be determined by the number of mtDNA molecules contributed by each parental gamete, and is expected to be highly variable among different taxa due to interspecific differences in mating systems and gamete investment. Estimates of this ratio are currently limited to few mammalian species, and data from other taxa are therefore needed to better understand the mechanisms of mitochondrial inheritance. The present study estimates mtDNA content in salmon sperm, the first nonmammalian vertebrate to be examined. Although highly divergent, it appears that the mtDNA content may be conserved within vertebrate taxa, indicating that the reduction of mtDNA is a key factor of spermatogenesis to ensure mitochondrial functionality on the one hand, and to avoid paternal leakage at a significant or detectable level on the other hand. We employ quantitative real-time PCR (Q-PCR) and demonstrate the accuracy and high reproducibility of our experiments. Furthermore, we compare and evaluate two standard approaches used for the quantification of genes, Q-PCR and blotting methods, in regard to their utility in the accurate quantification of mitochondrial genes.