Background <p>Global freshwater fishes face multiple pressures, including habitat degradation, overfishing, and environmental pollution, leading to a continuous decline in genetic diversity. Ussuri catfish (<i>Pseudobagrus ussuriensis</i>, Chinese common names: Huangziang and Niuweiba) is an endemic Chinese species of considerable ecological and economic importance. Declines in population size and degradation of wild stocks have raised significant conservation and aquaculture concerns. To promote the effective conservation and sustainable use of this species’ germplasm resources, we analyzed the genetic diversity and population structure of 149 individuals from three wild populations and two cultured populations using mitochondrial genes (<i>Cytb</i> and <i>COI</i>) and genotyping-by-sequencing (GBS) data. These results provide a scientific basis for artificial breeding, stock enhancement, and local germplasm conservation.</p> Results <p>Mitochondrial analyses revealed higher genetic diversity in wild populations compared to cultured populations, with maternal lineages being relatively conserved; however, the QH and HTH populations harbored region-specific haplotypes. Population differentiation analysis indicated that the HTH population exhibits a relatively independent genetic structure. Inter-population variation based on <i>Cytb</i> and <i>COI</i> was 51.40% and 55.33%, respectively, whereas intra-population variation was relatively low. Neutrality tests suggested a recent expansion in the HLJ population, while the HTH population may have been influenced by balancing selection or a bottleneck effect. GBS sequencing yielded a total of 1,238,359 SNPs, of which 168,794 high-quality loci were retained after stringent filtering. Some SNPs (e.g., contig15925, contig70883) displayed significant allele frequency differences among populations, representing potential markers of population differentiation. The average polymorphic information content (PIC) of SNPs and InDels was low, at 0.154 and 0.147, respectively, and average nucleotide diversity (π) was 0.287 and 0.273. Most loci conformed to Hardy–Weinberg equilibrium. Genetic structure analyses revealed pronounced differentiation among populations, with the QH population exhibiting the most distinct genetic features, followed by HTH. ADMIXTURE analysis further showed that HLJ, YJ, and SC populations shared similar ancestral components, whereas HTH and QH retained relatively independent genomic signatures. Isolation-by-distance analysis indicated no significant correlation between genetic and geographic distances.</p> Conclusion <p>Distinct populations of <i>P. ussuriensis</i> exhibit pronounced genetic differentiation and region-specific haplotypes, with wild populations showing higher genetic diversity than cultured populations. We recommend prioritizing in situ conservation and germplasm purification of wild populations, and using local genetic resources preferentially in stock enhancement programs, in order to maintain genetic diversity and ensure sustainable utilization.</p>