Abstract: To develop codominant and polymorphic microsatellite markers for Paracanthobrama guichenoti and to conduct genetic monitoring of its natural populations for the purpose of germplasm conservation and sustainable utilization, we characterized microsatellite sequences (2–6 nucleotide repeats) in the genome, screened polymorphic microsatellite markers, and assessed the genetic variation of three wild populations from Bali Lake (BL), Junshan Lake (JL), and Daguan Lake (DL) in the mid-lower Yangtze River basin. A total of 771,961 perfect microsatellite sequences were identified from P. guichenoti genome, with dinucleotide repeats being the most abundant (67.34%), followed by tetranucleotide repeats (22.45%). The 23 markers revealed a total of 213 alleles across the three populations. The polymorphism information content (PIC) per locus varied from 0.661 to 0.919, and all markers were highly polymorphic (PIC≥0.5). The values for the average number of alleles per population (N_o=5.913–8.565), observed heterozygosity (H_o=0.448–0.819), and PIC (PIC=0.579–0.786) revealed that all three populations were high polymorphic (PIC>0.5). These results suggest that P. guichenoti populations in the mid-lower Yangtze River lake region possess a relatively high level of genetic diversity. Analysis of molecular variance (AMOVA) indicates that 88.2% of genetic variation occurred within populations, while 11.8% occurred among populations. The genetic differentiation coefficient (F_st) among populations was 0.118, suggesting moderate genetic differentiation. Frequent gene flow (N_m) was observed between BL and JL populations (N_m=5.985>4), while the genetic differentiation (F_st) between these two populations and DL population reached a moderate to high level (0.05<F_st<0.25). It is hypothesized that differences in the founder effect, resulting from variations in river-lake connectivity, are the key factors influencing population genetic diversity and differentiation.