Genetic diversity in mtDNA was assessed within the unisexual (all female) hybridogenetic fish Poeciliopsis monacha-occidentalis and the two sexual species from which it arose. Results confirm that P. monacha was the maternal ancestor and that paternal leakage of P. occidentalis mtDNA has not occurred. Of particular interest is the high level of de novo mutational divergence within one hybridogenetic lineage that on the basis of independent zoogeographic considerations, protein electrophoretic data, and tissue grafting analysis is of monophyletic (single hybridization) origin. Using a conventional mtDNA clock calibration, we estimate that this unisexual clade might be >100,000 generations old. Contrary to conventional belief, this result shows that some unisexual vertebrate lineages can achieve a substantial evolutionary age.