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The sterlet (Acipenser ruthenus Linnaeus, 1758) is a relatively small sturgeon widely distributed in Eurasian rivers from the Danube to the Yenisei. During the twentieth century, all wild sterlet populations have declined due to anthropogenic factors including: overfishing, poaching, construction of dams, and pollution. Despite the necessity of characterization both wild and captive stocks, few studies of population genetics have been performed thus far. Here we studied the genetic diversity and geographic structure of sterlet populations across the eastern range – Ob-Irtysh and Yenisei basins – by sequencing a 628-bp fragment of mitochondrial DNA control region. We identified 98 new haplotypes, delineated 12 haplogroups and estimated the time of basal haplogroup divergence within the species as over 8 million years ago. Our data suggest that Ob-Irtysh and Yenisei populations are isolated from each other and much lower genetic diversity is present in the Yenisei population than in the Ob-Irtysh population. Our data imply that sterlet populations in Siberian rivers underwent bottleneck or fragmentation, followed by subsequent population expansion. The data obtained here are important for sterlet population monitoring and restocking management.