Abstract: River-bed and various substrata provide diverse habitats for fish. With the purpose of revealing varies of fish assemblage which caused by changes of bottom substrate after river realignment, the difference of fish communities were compared before and after the river regulation in two typical habitats of the upstream Changjiang in 2017 and 2019, and the two habitats are the fast flowing riffle (depth<0.3 m, velocity>0.3 m/s) and slow flowing river (depth<0.4 m, velocity<0.3 m/s). The result indicates that there is no significant difference (P>0.05) in the number of fish species between the two surveys, though fishes in collection decreased from 24 to 17 species after the river realignment. However, fish community diversity and evenness are decreased significantly after the river realignment (P<0.05) except for the Marglef richness index in spring and summer. The values of Shannon-Weiner index, Simpson index, Margalef richness index and Pielou evenness index were 1.623-2.253, 0.715-0.866, 1.680-3.109, 0.652-0.813 before the river regulation, 1.120-1.679, 0.528-0.724, 1.444-2.695, 0.508-0.722 after the river regulation, respectively. It was also found that the diameter of substrate on the riverbed decreased significantly (P<0.05), and so did the diversity of particle size. We speculated that the variations of fish community can be attributed to the decrease of habitat heterogeneity which is closely associated with riverbed structure. Therefore, to maintain the habitat heterogeneity, the selective removal of big size river-bed substrate should be avoided in the process of river realignment.