White sturgeon (Acipenser transmontanus) in the Fraser River are listed as imperiled (the second highest possible rating) by the British Columbia Conservation Data Centre. A difficulty in trying to protect this species in the Fraser River and elsewhere is the lack of knowledge regarding their population biology. Variation in the mitochondrial DNA control region and at four microsatellite loci was examined in order to characterize white sturgeon samples from throughout the Fraser River mainstem and from a major tributary, the Nechako River. Samples from the adjacent Columbia River were analyzed for comparison. In contrast to previous work, present data indicate that white sturgeon population structure in this region reflects post-glacial dispersal more than it does recent anthropogenic effects. The data divided the Fraser into four biogeographic regions: (i) the lower Fraser, below Hell’s Gate; (ii) the middle Fraser, between Hell’s Gate and river km 553; (iii) the upper Fraser, above the Nechako confluence; and (iv) the Nechako River. These four groups are concordant with those suggested by tag and recapture and catch per unit effort data, and are separated by what have been identified as barriers to white sturgeon migration. Based on concordance between these different types of data, it is argued that the four groups identified here merit evolutionarily significant unit (ESU) status.