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In 2009–2018, the isotopic composition of oxygen and hydrogen in the atmospheric precipitation, groundwater and river and lake water of Lake Onega basin was studied. The weighted annual isotope composition of precipitation at Petrozavodsk was δ¹⁸O = −11.7‰ and δ²H = −84‰ and varied from −30.9 to −4.1‰ for δ¹⁸O and from −23 to −22‰ for δ²H. The isotopic composition of the water in Lake Onega was relatively uniform from −11.5 to −9.3‰ for δ¹⁸O and from −85 to −71‰ for δ²H. In the bays, the isotopic composition of the water varied more substantially than in the central part of the lake due to the river runoff during springtime flooding. In late summer, the concentrations of deuterium and oxygen-18 increased in the lake water, and figurative points on the δ²H vs. δ¹⁸O diagram shifted above the meteoric line. The absorption of the isotopically heavy summer precipitation and disequilibrium isotope fractionation during evaporation led to the enrichment of the lake water by heavy isotopes. Experiments were conducted to estimate the evaporation influence on the isotope enrichment of the residual water, and a comparison of the obtained isotope data with the experimental function showed that commonly, about 4% and up to 12% of water was lost during the spring and summer, respectively. In the water of the tributaries, the abundance of the deuterium and oxygen-18 varied in a wider scale than in the lakes, from −14.4 to −9.1‰ for δ¹⁸O and from −102 to −73‰ for δ²H. An evaporation loss of up to 35% was found for the rivers in late summer, and this value was proportional to the area of lakes and wetlands in the elementary watershed. The initial isotope composition of the water in the tributaries prior to evaporation was estimated to be δ¹⁸O ≈ −14.1‰ and δ²H ≈ −103‰ on average and crossed the approximation and meteoric lines. This estimation was close to the average composition of the groundwater, i.e., δ¹⁸O ≈ −13.4‰ and δ²H ≈ −94‰ on the Lake Onega catchment. The slightly increased isotope depletion of the calculated composition in the initial river water in comparison with the groundwater was the result of the contribution of the spring snowmelt water, which had a significant influence on the lake water balance.