The percid genus Stizostedion consists of five species, three in Europe and two in North America, and is believed to have evolved in Europe and then later colonized North America. Three hypotheses exist concerning the route and timing of this colonization: a Miocene Atlantic "riverine" route; a Pliocene Beringian route; and a late-Pleistocene Atlantic route. To test these hypotheses, allozyme and mitochondrial DNA variation were compared in three Stizostedion species, S. canadense and S. vitreum from North America, and S. lucioperca from Europe, to determine their genetic relatedness. Divergence times between S. canadense and S. vitreum were estimated as 3.12 ± 1.33 million years before present (MYBP) from allozyme data and 4.06 ± 0.73 MYBP for mtDNA data, while divergence times between the North American species and S. lucioperca were 10.59 ± 2.74 MYBP and 7.86 ± 1.18 MYBP, respectively. These results are concordant with the hypothesis that colonization of North America by Stizostedion occurred during the Pliocene via Beringia.