C₂₅ highly branched isoprenoids (HBIs) are generally regarded as biomarkers signaling diatoms and have been widely detected in lakes in the lower-middle reaches of the Yangtze River recently. The lakes in Yunnan Provinces are rich and diverse in diatoms, whereas the reports on C₂₅HBIs still very limit. In this study, we first detected C_25:1HBI and C_25:2HBI in sediments of Lake Erhai, and further revealed the potential biogeochemical significance of C₂₅HBIs by comparing the spatial differences of the proxy and other underlying factors such as TOC, TN, water depth, etc. The isomers of both C_25:1HBI and C_25:2HBI were not detected in all surface sediments in Lake Erhai. The concentration of C₂₅HBIs was 11.18 μg/g(TOC), which was much higher than those in eutrophic Lake Taihu and similar with those in Lake Chenghu. The concentration of C_25:1HBI increased from the north to the south in Lake Erhai, while inverse trends for the concentration of C_25:2HBI. C_25:1HBI in Lake Erhai might come from certain predominant diatoms in the southern lake. In sediments with lower concentration of C_25:2HBI (<0.4 μg/g), there existed a weak positive correlation between C_25:1HBI concentration and C_25:2HBI concentration, while in some shallow areas, the concentration of C_25:2HBI was high (>0.4 μg/g) and the concentration of C_25:1HBI was low. Besides, the correlation between C₂₅HBIs concentration and TOC/TN displayed distinct spatial differences. In the northern part of Lake Erhai, C_25:1HBI-sourced diatoms might be inhibited by a large amount of nitrogen and phosphate fertilizer inputs related to agricultural activities. In the central part of Lake Erhai, the concentration of C₂₅HBIs showed a weak positive correlation with TN, but no correlation with TOC, indicating that the underlying control factors of the C₂₅HBIs-sourced diatoms were relatively complex. In the southern part of Lake Erhai, a strong positive correlation could be observed between C_25:1HBI concentration and TN, and between C_25:1HBI concentration and TOC. But the correlations between C_25:2HBI concentration and TN, and between C_25:2HBI concentration and TOC were weak. The presence of C_25:1HBI in the southern part of Lake Erhai could track eutrophication conditions, which was different from those observed in the middle and lower reaches of the Yangtze River. Meanwhile, C_25:2HBI might be contributed from the diatoms also producing C_25:1HBI and other diatoms adapted to live in shallow water, so C_25:2HBI was strongly affected by the hydrological conditions.