Abstract: Community assembly mechanisms have long been a central issue in ecology. Three mechanisms have been proposed to govern community assembly: neutral process, competitive interaction, and environmental filtering. To explore the assembly mechanism of fish communities in the middle reaches of the Yangtze River, we collected fish samples from 5 reaches (Yichang, Zhijiang, Jingzhou, Hannan, Hukou) and conducted a series of analysis including phylogenetic community structure analysis. The results showed that: (1) spatial clustering analysis revealed that all sampling reaches were identified as 3 groups (Yichang, Zhijiang + Jingzhou, Hannan + Hukou) with a similarity level of 65% or 2 groups (Yichang, Zhijiang + Jingzhou + Hannan + Hukou) with the similarity level of 55%. These groupings were consistent with the spatial distribution of the sampling reaches; (2) The assembly mechanisms of fish communities were distinct at different spatial scales. At local scale, competitive interaction drove the fish community assembly in Jingzhou sampling reach, while environmental filtering drove the fish communities assembly in other sampling reaches. At regional scale, environmental filtering structured the fish community in Yichang sampling reach, while interspecific competition structured the fish communities in the other 4 sampling reaches. Therefore, the local environment and spatial scales play a role simultaneously during fish community assembly in the middle reaches of the Yangtze River. The fish community in Yichang reach is structured by environmental filtering, which may be due to the swift current there. In other reaches, environmental filtering drove the fish community assembly at local scale, while competitive interaction played key role at regional scale. This may be because as spatial heterogeneity increases, distantly related species were contained in the community assemblage. This phenomenon is different from those in terrestrial plant communities, where the assembly mechanism has changed from small-scale competitive interaction to large-scale environmental filtering.