Abstract: After the implementation of 10-year ban fishing in the Yangtze River, the rare and endangered fish species Ochetobius elongatus has reappeared in the middle reaches of the Yangtze River. However, the genetic diversity and population structure of O. elongatus in this region remain unclear, hampering the development of effective conservation strategies. In this study, two mitochondrial genes and two nuclear gene sequences were used as molecular markers to analyze the genetic diversity, genetic differentiation, and population historical dynamics of four O. elongatus populations: the Hanjiang River, South Dongting Lake, Ouchi River, and the Gongan section of the Yangtze River. The results showed that haplotype diversity of O. elongatus in the middle reaches of the Yangtze River was high, the nucleotide diversity was low, and the overall genetic diversity was at a medium level. Molecular variance analysis (AMOVA) and genetic differentiation index (F_ST) showed that there was no significant genetic differentiation among O. elongatus populations in the middle reaches of the Yangtze River. The analysis of population historical dynamics indicated a possible population expansion of O. elongatus in the middle reaches of the Yangtze River approximately 165400 years ago, during the warm interglacial period between Lushan Glacial and Dali glacial. Furthermore, clear differentiation was observed between O. elongatus populations in the middle reaches of the Yangtze River and those in the middle and lower reaches of the Xijiang River, with an estimated divergence time was about 55500 years ago. Based on these findings, we propose considering O. elongatus populations in these two river basins as distinct conservation units when formulating conservation strategies.