Poecilia formosa, a small live-bearing fish native to northeastern Mexico, was the first recognized vertebrate with unisexual reproduction (Hubbs and Hubbs, 1932). This all-female "species" produces diploid apomictic eggs (Rasch et al., 1982; Monaco et al., 1984), and embryogenesis is subsequently activated by sperm from a related bisexual species, normally either P. latipinna or P. mexicana (Turner et al., 1980a, 1980b). The process apparently takes place without syngamy, such that genetic transmission is predominantly or exclusively clonal (Balsano et al., 1989). The gynogen P. formosa almost certainly arose via hybridization of P. latipinna and P. mexicana (Hubbs and Hubbs, 1932; Turner, 1982). It is sympatric with these species (Darnell and Abramoff, 1968), possesses an intermediate morphology (Hubbs and Hubbs, 1932; Meyer, 1938), and exhibits nearly fixed heterozygosity at numerous protein and allozyme loci that distinguish or are polymorphic in P. latipinna and P. mexicana (Abramoffet al., 1968; Balsano et al., 1972; Simanek, 1978; Turner, 1982). There is a relative paucity of allozyme variation among the unisexuals, and the best fit to the particular P. formosa alleles occurs in populations of P. mexicana in the Rio Tigre drainage near Tampico, Mexico. This led Turner (1982) to hypothesize that the gynogen may have arisen recently in this area (perhaps from a single hybridization event), and that its current geographic distribution reflects a rapid northward expansion via efficient colonizing ability. A relatively recent origin for P. formosa is also consistent with the finding that the species retains a capacity to express male-specific genes and to undergo normal spermatogenesis upon experimental induction with exogenous androgens (Turner and Steeves, 1989). One open evolutionary question concerns the direction of the cross that produced P. formosa. Unlike the situation in hybridogenetic Poeciliopsis fishes from western Mexico, where results of laboratory hybridization experiments have been critical in deciphering the male and female parents of the unisexuals (Schultz, 1973, 1989; Wetherington et al., 1989), attempts to synthesize unisexual P. formosa through laboratory crosses thus far have failed (Turner, 1982). Balsano et al. (1989) suspect that P. mexicana was the female parent, but note that direct evidence from maternally transmitted mitochondrial DNA (mtDNA) might settle the issue. Here we survey mtDNA genotypes in these Poecilia fishes, and show unequivocally that P. mexicana was indeed a recent female parent of P. formosa.