Phoxinus eos-neogaeus unisexual hybrids (Cyprinidae, Pisces) are among the few vertebrate taxa known to reproduce clonally by gynogenesis. These taxa have a broad distribution in North America, mostly located in regions previously covered by the last Pleistocene ice sheet. To assess whether asexual hybrids dispersed from glacial refuges at the end of the Pleistocene or they originated from current hybridization events, genetic diversity of mitochondrial DNA (mtDNA) sequences and microsatellite loci was determined in populations from 16 different sites in the Mississippi–Missouri River (Nebraska and Montana), Rainy River–Hudson Bay (Minnesota), and St Lawrence River (Quebec) drainages. The maternal species (P. neogaeus) occurred in Minnesota and Nebraska but was absent from Montana sites and was restricted to only two of 11 lakes sampled in Quebec, although hybrids were present at all sites. The genetic survey revealed a total of 49 clones, originating from 14 hybridization events. Several of the lineages were characterized by mtDNA haplotypes not detected in the maternal ancestor. Lineages as well as clones frequently displayed a large geographical distribution at a regional scale. Dating of hybridization events suggested a relatively recent origin (< 50 000 years ago) from the Mississippi glacial refuge, even in regions not covered by the last Pleistocene glacier. Altogether, these results indicate P. eos-neogaeus hybrids are not the result of current hybridization events, but display a pattern predicted by postglacial dispersal. Our findings have considerable implications for the nature of selection processes affecting the diversity of these asexual taxa and their coexistence with sexual ancestors.