Most of the northern half of North America’s freshwater fish survived the last glacial period in unfrozen refugia peripheral to the ice sheets. In our study, the question of which refugia Arctic grayling (Thymallus arcticus) inhabited during the Wisconsinan Ice Age, and how they subsequently dispersed to their present geographical range, was examined using mitochondrial DNA (mtDNA) analysis. mtDNA from 12 T. arcticus populations was analysed by direct sequencing and restriction fragment length polymorphism analysis (RFLP). Our data support the hypotheses that T. arcticus had a large refugial population in the Bering Refuge (shown by high mtDNA diversity in extant Alaskan populations) and that British Columbia was colonized from the Bering Refuge (shown by mtDNA haplotype similarities). Our data also show that a disjunct southern set of populations in Montana is significantly different from the northern grayling, in terms of restriction haplotype frequency and distinguishing sequence characteristics. Sequence results yielded an estimated divergence time of 370 000 years between the northern and Montana grayling haplotypes. We conclude that T. arcticus survived the Wisconsinan glaciation in at least two refugia: (i) the Bering Refuge north of the ice sheets; and (ii) either the Upper Missouri or the southwest Alberta Refuge, south of the ice sheet.