The effects of Quaternary glacial range partitioning on the diversification of Holarctic biota remain unclear. Glacial refugial lineages may form vicariant species, hybrid products, or merge after secondary contact. Here, we assess the effects of Quaternary glaciation on a Holarctic sexual zooplankter, Daphnia galeata, with apparently marked dispersal capacity and a widespread hybrid lineage in the New World. We collected samples of this species from 148 Holarctic lakes, analysed the nuclear and mitochondrial gene sequences, and tested predictions for hypotheses that account for the origin and spread of the New World D. galeata. We detected five nuclear phylogroups and four mitochondrial phylogroups, most of which were restricted to either the New World or the Old World. The oldest mitochondrial phylogroup was restricted to Japan. One major mitochondrial clade was distributed throughout the Holarctic, but only four haplotypes were shared among continents, and analysis of molecular variance indicated significant structure at the continental level. Haplotype sharing among continents could largely be attributed to anthropogenic introductions. Mismatch distributions, haplotype networks, phylogenetic trees, longitudinal haplotype diversity erosion and coalescence analyses are consistent with colonization from an Old World and a New World refugium. Our nuclear and mitochondrial DNA sequence evidence supports the hypothesis that the New World D. galeata underwent introgression with Daphnia dentifera, with dispersal being enhanced by glaciation. We conclude that Quaternary glaciation had a pronounced effect on the diversification of a Holarctic sexual zooplankter.