The Yangtze River drainage in China is among the most species rich rivers for freshwater mussels (order Unionida) on Earth with at least 68 species known. The freshwater mussels of the Yangtze River face a variety of threats with indications that species are declining in abundance and area of occupancy. This study represents the first analyses of the genetic structure and diversity for the common and widespread freshwater mussel Nodularia douglasiae based on microsatellite DNA genotypes and mitochondrial DNA sequences. Phylogenetic analysis a fragment of the COI mitochondrial gene indicated that N. douglasiae collected from across the middle and lower Yangtze River drainage are monophyletic with N. douglasiae from Japan, Russia, and South Korea. The results of the analysis of both the mtDNA and microsatellite datasets indicated that the seven collection locations of N. douglasiae in the middle and lower Yangtze River drainage showed high genetic diversity, significant genetic differentiation and genetic structure, and stable population dynamics over time. Moreover, we found that the connections among tributaries rivers and lakes in the Yangtze River drainage were important in maintaining gene flow among locations that N. douglasiae inhabits. An understanding of the genetic structure and diversity of a widespread species like N. douglasiae could be used as a surrogate to better understand the populations of other freshwater mussel species that are more rare in the Yangtze River drainage. At the same time, these results could provide a basis for the protection of genetic diversity and management of unionid mussels diversity and other aquatic organisms in the system.