In eastern North America there are populations of all-female salamanders that incorporate the nuclear genomes of two or three of four sympatric bisexual species. The hybrids can be diploid, triploid, tetraploid or pentaploid, and 18 different combinations have been reported. All hybrids require sperm from a sympatric male of one of the bisexual species to reproduce, but the sperm may or may not be incorporated in the egg. Some of the hybrids are believed to represent separate, clonal species, but little is known of the origin of this hybrid complex. Vertebrate mitochondrial DNA is inherited maternally, allowing identification of the female parent that gave rise to hybrid lineages. A portion of the cytochrome b gene was sequenced from diploid and triploid hybrids that represent combinations of all four species. Nearly all hybrids had a similar mitochondrial genome sequence, independent of nuclear genome composition and ploidy, and the sequence was distinct from that of any of the four bisexual species. The hybrids maintain a mitochondrial lineage that has evolved independently of their nuclear genome and represent the most ancient known unisexual vertebrate lineage.