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The analysis of biodiversity spatial patterns and the detection of hotspots are effective ways for conservation planning and scientific management of biodiversity. Based on field investigation, historical data and literature information, we constructed the comprehensive index of biodiversity by considering habitat quality, species diversity and landscape diversity within Chongqing Pengxi River Wetland Nature Reserve. Combined with spatial autocorrelation analysis, we analyzed spatial distribution pattern and the degree of spatial autocorrelation of biodiversity in the nature reserve, identified biodiversity hotspots, and discussed the protection effectiveness of existing nature reserve for the hotspots. The results showed that the spatial pattern of biodiversity in the nature reserve tended to decrease with the increases of distance from the river and riparian zone. Areas with high value of biodiversity index were mainly concentrated in Pengxi River, Puli River, Baijiaxi Stream and alongshore areas. There was significant positive correlation in the spatial distribution of biodiversity. The spatial autocorrelation of local areas was mainly characterized by high-high aggregation and low-low aggregation. The area of biodiversity hotspots was 457 hm 2 , accounting for 11.1% of the total nature reserve. The core area of current nature reserve covered 51% of the hotspots and 50% of the secondary hotspots. The structure and functional zone distribution of nature reserve should be further optimized and adjusted. We suggested that other hotspots should become part of the core area, including the Longwangtang area of Puli River, Xiaoyakou, Dengjiawan, Dongziyan, Longwangtang, Jiuwuju, Huatouju, Xinpuzi and Longjia yard of Baijiaxi Stream. The cold spots should be removed from the core area, to improve the function zoning of nature reserve. Our results could provide quantitative basic references for the boundary optimization and control zoning of nature reserve, and the reasonable promotion of the adjustment of "three zones to two zones", and could have guiding significance for improving effectiveness of species conservation and formulating scientific protection strategies.