Microbial antioxidant functions hold significant application potential in the development of holistic health resources, yet their spatial distribution patterns across continuous ecological gradients remain poorly understood. [Objective] To analyze the diversity and spatial distribution characteristics of antioxidant function-related genes in prokaryotes within the Lasha Mountain watershed. [Methods] A spatially exhaustive nine-grid sampling strategy was employed, with 117 sampling points established across the watershed and its 12 sub-basins. PICRUSt2 was used for functional prediction based on high-throughput sequencing data of the 16S rRNA gene. [Results] A total of 38 genes associated with antioxidant compound synthesis were identified and categorized into 12 functional groups. The diversity and total abundance of antioxidant function-related genes were unevenly distributed within the Lasha Mountain watershed, with the downstream region exhibiting higher gene diversity and abundance. The genes associated with non-enzymatic antioxidant compounds dominated both in functional category richness (7 categories) and abundance (0.005 270-0.007 657), with glutathione and thioredoxin genes exhibiting the highest abundance. The overall abundance variation of individual genes (CV=125.85) primarily originated within sub-basins (CV=77.23). After the antioxidant function-related genes were classified by synthesized compounds, the pattern of variation reversed. [Conclusion] The antioxidant function-related genes of prokaryotes exhibit spatial distribution differences in the watershed, and the downstream area—characterized by multiple coupled stress conditions—is a hotspot for the diversity and total abundance of such genes. This study provides an ecological basis for the targeted screening of high-quality antioxidant strains.