The East Asian monsoon and related seasonal rain belts are assumed to be significantly variable at intraseasonal, interannual, and interdecadal time scales, and largely determine the hydrology and ecological processes of the streams and rivers in this region. This study investigated the spatiotemporal variation in fish assemblages and the relative importance of local habitat and regional landscape variables in two protected streams in the middle Yangtze River basin. We hypothesized that the stream fish assemblages in this area are strongly influenced by the monsoon climate, and display a spatiotemporal variation in response to changes of stream flow regimes and physical habitats. The results show that species richness and abundance varied with both season and stream size. The distance to the downstream confluence site was the top important environmental factor, followed by altitude and downstream link. The results suggest that the fish assemblages within this protected watershed are mainly affected by regional landscape variables such as longitudinal position and stream position, but less by local habitat variables including water temperature and habitat types. The study highlights the importance of tributary connectivity and continuity to stream fish diversity in this area, indicating that restoration activities should preferentially recover the connectivity between upstream and downstream.