Abstract: Lake Dongting is the second largest floodplain lake along the Yangtze River basin. However, the study on fish communities in the Dongting Lake is limited, lacking systematic research on the spatio-temporal variations of fish assemblages in this area. Fish were sampled seasonally from December 2012 to October 2014 in the Dongting Lake. 80 fish species belonging to 54 genera, 17 families, and 7 orders with dominant Cyprinidae accounting for 58.8% of the total species were collected. The species richness reduced by 36 in contrast with the historic records. The number and percentage of riverine and river-sea migratory species decreased by 13 (2.2%) and 4 (3.0%) respectively in comparison with historic records. We detected significant seasonal and regional differences in species composition and biodiversity. The diversity and evenness of fishes were significantly higher in summer and autumn than those in other seasons. Meanwhile, significantly lower diversity was observed in the east Dongting Lake than other regions. Our results indicated that seasonal flood pulses and, assisting by river-lake connectivity may play a fundamental role for the fish resources supplement in floodplain lakes. This study demonstrated significant spatio-temporal variations of fish assemblages in the Dongting Lake, which provided scientific foundations for fish conservation in this area.