The Yangtze River floodplain is an area with an extremely rich diversity of fish species. Poyang Lake, as an important part of this river–floodplain system, is a crucial habitat for the survival of fish. However, prolonged human activities, such as environmental pollution and hydroelectric development, have degraded the habitat in the Poyang Lake Basin, posing threats to fish populations. Understanding genetic diversity is crucial for maintaining fish populations and understanding their dynamics. The genetic diversity of Hemiculterella wui, an economically endemic species in China of subfamily Cultrinae (Cypriniformes: Cyprinidae), has been understudied. This study investigated the genetic diversity and structure of H. wui populations from the Ganjiang, Xinjiang, Fuhe, and Raohe rivers in the Poyang Lake Basin using mitochondrial Cytb gene analysis. Results showed high haplotype diversity but low nucleotide diversity in H. wui’s Cytb sequences. Analysis of molecular variance (AMOVA) showed no significant geographic genetic structure among populations. Haplotype network analysis revealed no clear geographical clustering. Neutrality tests and haplotype nucleotide mismatch distribution indicated that all populations had experienced expansion events. These findings suggest that H. wui in Poyang Lake does not show a distinct geographic structure. However, it is still necessary to monitor the genetic characteristics of H. wui to maintain the genetic diversity of fishes in Poyang Basin, considering the threat of habitat loss and fragmentation to the population.