Abstract: To better protect the fish resources in the Xijiang Rare Fish Provincial Nature Reserve, we used the environmental DNA (eDNA) technology to analyze the fish species composition and fish diversity at different cross sections of 18 sampling stations on the left, middle and right banks of the river section. Two molecular markers, i.e. COI and 12S rRNA, were used in this study. The results show that the two genes of 12S rRNA and COI were detected in 53 fish species, belonging to 48 genera, 12 families and 4 orders. Forty-seven and nineteen fish species were examined based on 12S rRNA and COI, respectively. In addition, 13 shared fish species were discovered by using the two gene markers. Alpha diversity analysis based on sequence abundance shows that the two markers unanimously supported the highest number of fish species and the highest richness of Chao 1 index on the left bank of the river section, indicating that the left bank appears to have higher fish diversity than the middle and right banks. Beta diversity analysis based on 12S rRNA shows obvious differences in the fish composition on the left, middle and right banks. The research provides basic data and technical support for the protection and dynamic monitoring of fish biodiversity in this nature reserve.