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This dataset comprises key water quality parameters collected from three Gamo Highland lakes (Donie Ela, Ayalew, and Ambassa) in Southern Ethiopia. The data were generated to evaluate the effects of seasonal variation, altitude, and anthropogenic impacts on the water quality of the studied lakes. Water samples were collected during two distinct seasons: the dry season (December 2024 to January 2025) and the rainy season (June to August 2024), from sites with high anthropogenic impact and from relatively less human-affected areas. Key physicochemical parameters, including dissolved oxygen (DO), electrical conductivity (EC), pH, temperature, total dissolved solids (TDS), and chlorophyll-a, were measured in situ using calibrated portable meters. Water transparency was determined using a standard 20 cm diameter black-and-white Secchi disk to measure Secchi depth (ZSD). For laboratory analysis, water samples were collected in acid-washed, pre-cleaned, and dried bottles. The samples were properly labeled, stored in ice boxes at approximately +4°C, and transported to the laboratory under controlled conditions. Laboratory analyses were conducted at the Limnology Laboratory of Arba Minch University. The analyzed parameters included ammonia (NH₃), nitrate (NO₃⁻), total phosphorus (TP), biological oxygen demand (BOD), chemical oxygen demand (COD), alkalinity (as CaCO₃), and total hardness (TH), following standard limnological procedures. This dataset provides essential information for understanding nutrient dynamics, trophic status, and ecological conditions of highland lakes, and it can support further research in limnology, water resource management, and environmental monitoring.