Abstract: A total of 188 Xenocypris microlepis specimens were collected from five populations in the Xiangjiang River: Yongzhou (YZ), Hengyang (HY), Dongjiang Lake (DJH), Chaling (CL), and Liuyang (LY). Mitochondrial DNA D-loop sequences were used to analyze the genetic diversity and historical demography. Analysis of the 188 sequences revealed 37 variable sites and 24 haplotypes. Haplotype diversity (H_d), nucleotide diversity (P_i), and genetic distances among populations ranged from 0.755 to 0.911, 0.00277 to 0.00556, and 0.00467 to 0.00658, respectively. Among the 24 haplotypes, only Hap1 was shared across all five populations, and no phylogenetic structure was observed. Analysis of molecular variance (AMOVA) revealed that most genetic variation occurred within populations (90.48%), while only 9.52% occurring among populations. UPGMA clustering based on genetic distance grouped HY with LY and YZ with CL. F_st-based estimations indicated low genetic differentiation between HY and DJH and between HY and CL; moderate differentiation between DJH and YZ, DJH and CL, and between LY and the other four populations; and no differentiation between YZ and CL or between YZ and HY. A Mantel test showed no significant correlation between genetic differentiation and geographic distance. Neutrality tests and mismatch distribution analysis suggested indicated no evidence of historical population expansion or contraction in the Xiangjiang River, whereas Bayesian skyline plots indicated a declining trend over the past 250000 years in all populations except HY. These findings provide valuable insights for evaluating the effectiveness of artificial restocking and for guiding conservation and utilization of X. microlepis genetic resources in the Xiangjiang River basin.