Maintaining fish diversity is essential for environmental protection. To characterize the fish composition of the tributaries and mainstream in the upper reaches of the Ganjiang River, we identified seventy-five species of 15 families in 14 sampling sites. These data were analyzed using the inclusion index at the taxonomic level (TINCLi), the importance value index (IVI), the taxonomic diversity indices (Δ+ and Λ+), cluster analysis and non-metric multidimensional scaling (nMDS). The results showed that the most common and dominant order was the Cypriniformes, represented most frequently by Hemiculter leucisculus among the sample sites. Most fishes were omnivorous, mountain stream or settled fish that lay sinking or viscid eggs. Most sites showed a trend of more discrete distribution from high latitude to low latitude. According to the taxonomic diversity indices, the fish taxonomic composition in the upper reaches of Ganjiang River is uneven. The Bray-Curtis resemblance matrix and nMDS showed that the habitats of the Ganjiang River were divided into four areas. The results will provide information needed for freshwater fish resource protection in the upper reaches of the Ganjiang River.