RFLP analysis of the ND4–ND5 genes of the mtDNA genome in Daphnia middendorffiana and three closely allied species was used to investigate its origin and age. Populations of D. middendorffiana from arctic Canada were found to possess three distinct mtDNA lineages, only one of which appears unique to this species. The other two mtDNA lineages are either closely allied or identical to haplotypes in D. pulicaria, suggesting that it is the maternal parent of many clones of D. middendorffiana. Within D. pulicaria, mtDNA lineages have largely disjunct distributions, suggesting that populations of this species persisted in three glacial refugia (arctic, western, eastern) during the Pleistocene. Hybridizations between these refugial stocks and other species such as D. melanica and D. pulex likely generated many of the polyploid lineages of D. middendorffiana following the Wisconsinan glaciation. The presence of one unique mtDNA lineage in D. middendorffiana suggests that at least some of its clones are more ancient, but further studies are needed to rule out the possibility of their recent derivation from an as yet undetected sexual species. As a general result, this study suggests that polyploid cladocerans are unlikely to predate the Pleistocene.