The largefin longbarbel catfish (Hemibagrus macropterus), a freshwater species endemic to China with fundamental economic importance, requires investigation into its genetic structure for effective management. In this study, we employed mitochondrial cytochrome b (Cytb) gene sequences and 14 microsatellite loci to elucidate the genetic structure of 195 individuals across eight distinct populations. The Cytb analysis revealed a haplotype number (H) of 31, haplotype diversity (Hd) of 0.853, and nucleotide diversity (π) of 0.0127. Population neutrality tests indicated that Tajima's D (-0.59467) and Fu and Li's D* (0.56621) were not statistically significant, and the mismatch distribution exhibited a multimodal pattern. Microsatellite analysis revealed that the mean number of alleles (Na), observed heterozygosity (Ho), and polymorphic information content (PIC) across all loci were 18.500, 0.761, and 0.808, respectively. The UPGMA phylogram constructed based on genetic distance identified two distinct clusters, with paired Fst values ranging from 0.108 to 0.138. These results suggest that the largefin longbarbel catfish is in a state of dynamic equilibrium with high genetic diversity. Furthermore, there was significant genetic differentiation between the YB population and the other seven populations, indicating that the population in the upper reaches of the Yangtze River should be managed as a distinct unit.