Schizothorax o’connori is endemic to the Yarlung Tsangpo River on the Tibetan Plateau. We assessed the relative impacts of historical and contemporary factors in organizing genetic variation in S. o’connori populations using mitochondrial cytochrome b sequences. We analyzed 191 samples from 11 populations and identified 78 haplotypes. The phylogenetic analyses and analysis of molecular variance all supported the same conclusions of two well-differentiated east–west phylogroups, separated by the Tsangpo Great Gorge. The split between the two clades accounted for 58% of the genetic variance observed among the examined samples. Waterfalls as effective barriers played an important role in shaping the phylogeographical structure of this species. Analyses of migration rates revealed that upstream dispersal was limited crossing waterfalls. Our study revealed substantial spatial and temporal variation in the influence of landscape features on contemporary patterns of genetic structure in S. o’connori. Interglacial range expansions clearly left their mark on contemporary populations above the Tsangpo Great Gorge.