Identifying the distribution of species composition and their abundance of fishes is basic for the conservation and management of fish diversity. Based on the data collected from 57 stream segments within the Taihu Lake Basin during October 2013 and May 2014, we examined how the stream fish assemblages vary spatially and seasonally in this study area. A total of 5051 individuals representing 46 species were collected, among which Cyprinidae fishes are amounted to 26 species. Fish diversity in October was significantly higher than that in May, and fish assemblage structures also significantly differed seasonally. Significant variations among different subbasins were observed for both fish species diversity and assemblage structures. The Yanjiang and Zhaoge subbasins had relatively lower species diversity, while those in the Nanhe and Tiaoxi subbasins were relatively higher. Assemblage structures in the Huangpujiang subbasin showed significantly difference from those in the Zhaoge, Tiaoxi and Yanjiang subbasins. When the spatial variations in fish assemblages among ecoregions were considered, fish assemblage structures, not species diversity, showed significantly variation between two ecoregions at level-1; while both assemblage structures and species diversity differed significantly among four ecoregions at level-2. This among-ecoregion variations in assemblage structures resulted from the spatial distribution of some dominant fishes, such as Hemiculter leucisculus, Carassius auratus and Pseudobrama simoni, and some rare fishes, such as Zacco platypus, Phoxinus oxycephalus, Oryzias latipes sinensis and Gambusia affinis.