Spinibarbus sinensis is one of the most important freshwater fish species in the upper reaches of the Yangtze River. Unfortunately however, the stock of the wild population declined sharply in recent years. There is an urgent need to understand the population structure of the fish to aid development of conservation measures and allow for continued exploitation of this important species. We collected 288 individuals from three geographic areas: Jialingjiang River, Chishuihe River, tributaries of the upper reaches of Yangtze River, and Qingjiang River, a tributary of the middle Yangtze River. The morphological variation and biology of the three populations was investigated using traditional morphological (one-way analysis of variance, cluster analysis, principle component analysis, and discriminant analysis) and truss network analysis. The three populations of S. sineisis differed significantly(P<0.05) for 29 morphological proportional characters(e.g., TL/BL, FL/BL, BW/BL). A cluster analysis indicated that the morphological charactersof S.sineisis in Jialing River were most similar to S. sineisis in Chishui River. The principal component analysis yielded eight principal components with a cumulative contribution ratio of 62.93%. The discriminant analysis suggested that these three populations could be divided. The discriminant functions of the three populations of S. sineisis were established based on 16 morphological characters that differed significantly among the populations. The total accuracy of discrimination of S.sineisis from the three populations was 99.31%. Our results suggest that there are significant morphological differences among S.sinensis populations in the three sampling areas in the upper reaches of Yangtze River basin