The transformation of ecosystems influences the genetic diversity and evolution of aquatic organisms. However, the mechanisms by which the genetic structure of the Daphnia population responds to the changes in ecosystem remain unclear. We used the mitochondrial cytochrome oxidase subunit (CO1) and 12S genes and microsatellite markers to investigate the genetic structure and differentiation of the Daphnia similoides sinensis population in the sediments (core depth, 25 cm) of Lake Junshan, China. The construction of a lake embankment in 1958 changed the lake ecosystem from an open to a closed state, interrupting species exchange and thus leading to a continuous decrease in the genetic diversity of the D. similoides sinensis population during 1959–2010 (14-3 cm), although its genetic structure was stable based on the analysis of 14 microsatellite markers. However, after 2010, the genetic diversity indexes showed an increase in the genetic diversity of D. similoides sinensis; there was also a significant change in the genetic structure. The changes in the genetic diversity and structure of D. similoides sinensis significantly correlated with eutrophication. The results suggest that lake embankment can reduce the genetic diversity of D. similoides sinensis during the early stages. The D. similoides sinensis population accelerated genetic differentiation during the rapid lake eutrophication period due to continuing barrier effect. This study clearly reveals the effects mechanisms of the rapid enclosure of aquatic ecosystems on the species diversity and genetic differentiation of aquatic organisms.