This study aimed to characterize fish assemblage and evaluate environmental influence on fish distribution in the Chishui River, a protected tributary of the upper Yangtze River. Thirty-one sites regularly distributed in longitudinal profiles were sampled in April, 2007. Sixty-two fish species belonging to 3 orders, 8 families, and 52 genera were collected. Species richness and diversity significantly increased from upstream to downstream. Canonical correspondence analysis (CCA) highlighted five environmental variables (altitude, conductivity, dissolved oxygen, channel width and current velocity) significantly structuring fish assemblages in the Chishui River. Based on species distributions and fish-habitat relationships, conservation strategies were proposed for different reaches.