Freshwater mussels play a vital role in maintaining aquatic ecosystem stability and are effective bioindicators of environmental change. In the Yangtze River Basin, mussel populations have experienced significant declines due to habitat degradation, hydrological alterations, and human disturbances. Understanding the genetic diversity and population structure of dominant species such as Cristaria plicata is crucial for evaluating their adaptive potential and guiding conservation strategies. In this study, mitochondrial cox1 and Cytb genes were used to assess the genetic diversity and population structure of C. plicata from four representative river-connected lakes in the middle and lower Yangtze River Basin: Shijiu Lake (SJ), Qili Lake (QL), Poyang Lake (PY), and Dongting Lake (DT). A total of 120 individuals were collected across hydrologically connected sites, and 90 cox1 and 42 Cytb haplotypes were identified. Haplotype diversity (Hd) and nucleotide diversity (π) were high (cox1: Hd = 0.951, π = 0.03310; Cytb: Hd = 0.855, π = 0.02118), with the DT population showing the highest diversity. Pairwise Fst and AMOVA analyses revealed significant genetic differentiation between SJ and the DT and PY populations (P < 0.01), but not between SJ and QL, and most genetic variation occurred within populations (cox1: 78.01%; Cytb: 83.23%). Neutrality and mismatch distribution tests indicated recent population expansion in SJ and QL, while PY and DT populations appeared relatively stable. The haplotype network and phylogenetic tree suggested partial gene exchange among lakes but also revealed region-specific lineages shaped by limited connectivity. Overall, C. plicata populations in the four lakes exhibited a characteristic “high Hd–low π” pattern, implying historical bottlenecks followed by expansion. Interestingly, smaller lakes (SJ and QL) contained more haplotypes than larger lakes (PY and DT). This pattern likely results from stochastic genetic drift, bottleneck recovery, and microhabitat heterogeneity in small, semi-isolated systems, where periodic hydrological isolation and reconnection alter gene frequencies. In contrast, large lakes with greater hydrological connectivity, habitat diversity, and abundant host fish resources maintain higher overall genetic variation through continuous gene flow. The observed spatial differences demonstrate that lake size, water connectivity, and ecological heterogeneity are the principal drivers of genetic diversity and population structure in C. plicata. Anthropogenic disturbances and hydrological fragmentation further exacerbate local genetic differentiation, particularly in smaller lakes.In conclusion, this study provides comprehensive evidence that C. plicata populations in the Yangtze River lake–river system maintain high genetic diversity but exhibit spatially structured genetic patterns shaped by hydrological connectivity and lake characteristics. Preserving ecological connectivity, protecting host fish resources, and reducing habitat fragmentation are essential for maintaining genetic variation and ensuring the long-term stability of freshwater mussel populations in the Yangtze Basin.