The Chinese longsnout catfish (Leiocassis longirostris) is an economically important freshwater fish in China; however, its wild resources have declined dramatically in recent decades. Understanding the genetic structure of Chinese longsnout catfish populations is crucial to guide breeding programs and fishy restoration. In this study, 15 highly polymorphic microsatellite DNA loci were used to evaluate its genetic diversity and population structure. Chinese longsnout catfish populations show high genetic diversity; they do not show significant genetic differentiation or systematic geographic pattern of variation. From the upper to the lower reaches of the Yangtze River, the genetic diversity of Chinese longsnout catfish populations showed an increasing trend. The Gezhouba and Three Gorges dams, which physically divide the Yangtze River into upstream and mid-downstream sections, did not contribute to the genetic differentiation of Chinese longsnout catfish populations. Hence, the source of broodstock is not critical for within-river breeding programs and stock enhancement to restore the wild population. In addition, possible effects of dams on differentiation among populations are crucial and long-term evaluation is essential.