Abstract: To assess the germplasm status of Odontobutis sinensis, and provide a scientific basis for parental selection during large-scale propagation and new variety breeding, we performed whole-genome resequencing on 60 wild individuals from three river systems (Fushui-FS, Hanjiang-HJ, and Xiangjiang-XJ) in the middle reaches of the Yangtze River to analyze their genetic diversity and selective signatures. In total, 12 901 465 high-quality single nucleotide polymorphisms (SNPs) were identified and filtered, with a density ranging from 5.711 0 to 9.219 4 per kilobase. The results reveal that genetic diversity was low among all populations (Nucleotide diversity, π=0.0017–0.002 9; polymorphism information content, PIC=0.244 0–0.260 3; observed heterozygosity, H_o=0.151 9–0.287 3). All populations showed signs of inbreeding (Inbreeding coefficient, F_HOM=0.124 9–0.537 7), and the FS population was the most inbred (F_HOM=0.537 7; Ho=0.151 9). Population structure analysis indicated significant genetic differentiation (Fixation index, F_st=0.263 9–0.323 9) and limited gene flow (N_m=0.521 8–0.697 3) among populations. The populations clustered into three genetic lineages. The XJ and HJ populations showed relatively homogeneous genetic background, whereas the FS population displayed a more complex genetic composition. Selective sweep analysis identified significant enrichment of genes involved in immune-related pathways in the FS population, such as the complement and coagulation cascades, the NF-κB signaling pathway, and the phagosome. In conclusion, when conducting large-scale propagation and new variety breeding, the XJ and HJ populations with simple genetic background and low inbreeding levels should be preferentially selected as parental sources.