Abstract: To protect the genetic diversity of a critically endangered species Ochetobius elongatus, taking seven individuals of O. elongatus collected from the field investigation in the Yangtze River as samples, we sequenced two mitochondrial genes (Cytb and ND2) and two nuclear genes (mhy6 and RAG2) for the seven samples by Sanger sequencing technique. Combining with the published four gene sequences of 52 O. elongatus samples in the Pearl River, we explored the genetic structure of O. elongatus between the Pearl River and the Yangtze River, so as to provide scientific support for its conservation. We applied phylogenetic analyses, haplotype networks and divergence time estimation. Phylogenetic analyses and haplotype networks reveal that O. elongatus populations in the two rivers generated two deep and independent lineages, and formed private alleles at the nuclear gene level. The results suggest that O. elongatus populations in the two rivers evolved independently without agene flow. The rapid lifting of the Qinghai-Xizang Plateau during the middle Pleistocene might be an important factor that triggered the split of O. elongatus populations in the two rivers 0.38~0.76 million years ago (Ma). In view of the strictly geographical division of O. elongatus populations between the two rivers at both mitochondrial and nuclear gene levels, we suggest that these two populations should be regarded as two evolutionary significant units, and targeted strategies should be urgently put forward to manage and protect its resources.