Human disturbances can have severe environmental impacts on freshwater ecosystems. The main aim of this study was to detect the influences of physiochemical variables, land-cover characteristics, and river connectivity on fish assemblages in Lake Chaohu Basin, China. A cluster analysis of the river connectivity variables identified four groups of sites that characterized by significantly different connectivity gradients in local scale. These four groups of sites showed increasing connectivity from the upper reaches to the lower reaches. At the same time, among the four groups, the values of the environmental variables generally increased from the upper reaches with less human activity towards the lower reaches with more human activities. For instance, some main physiochemical variables (e.g., river width, water depth, nitrate, phosphate) significantly increased among the four groups. In contrast, fish taxa richness and diversity indices were not significantly different among the four connectivity groups. However, fish assemblages showed significant variation among the connectivity groups (p=0.026). In addition, the study determined that upper riparian land uses (e.g., woodland and grassland), flow velocity, and elevation were environmental variables regulating the variance in the fish communities, and for the connectivity variables, only river order and number of branches along a path to the left of the main stem affected the variance in the fish communities. Therefore, new practices aimed at maintaining and even increasing the riparian canopy coverage and the flow velocity of rivers should be integrated into local conservation planning for freshwater ecosystems, especially in the upper reaches of the basin.