Ecosystems of thermokarst lakes in Western Siberia perform significant climate-regulating functions in the biosphere, acting as indicators of ongoing climate change and possible increase in anthropogenic load. To better understand the processes of formation, nutrition, and water balance, we studied the oxygen and hydrogen isotopic composition (δ18O and δ2H) in pore waters of three thermokarst lakes with different surface areas. The analysis was performed by isotope equilibration mass spectrometry using a DELTA V Advantage isotope mass spectrometer (Thermo Fisher Scientific, Germany) and a GasBench II system. The δ values were calculated according to the accepted international standard sample of mean ocean water (Vienna-Standard Mean Ocean Water, IAEA, Austria). The values of δ18O and δ2H range from –10.8 to –11.8‰ and from –76.4 to –88.3‰, respectively. The lightest hydrogen isotopic composition (–88.3 ± 3.4‰) is found in a large lake over 0.5 km2. This is more than 8‰ different from the heavier composition for small and medium-sized lakes (–76.4 ± 3.9 and –72.7 ± 6.5‰, respectively). Deuterium excess values, reflecting the deviation of isotope values from the Global Meteoric Water Line (GMWL), show a noticeable difference among lakes of different sizes. This may indicate different processes of kinetic fractionation of the isotopic composition, such as evaporation. The magnitude of deuterium excess for a large lake is 3.6 ± 7.6‰. Thus, it can be concluded that precipitation (evaporation and snowmelt) has a great influence on the pore water isotopic composition of lakes with a large water mirror area. The pore water isotopic composition of a small lake (400 m2) has a mean deuterium excess value of 9.52‰, which fits within the GMWL and may reflect the predominance of atmospheric supply (rainwater). The highest fractional isotopic composition is characteristic of a medium-sized, actively growing lake with a deuterium excess of 21.8‰. The above-described differences in the isotopic composition of oxygen and hydrogen in the pore waters of the bottom sediments of the studied thermokarst lakes are probably the result of a combination of several factors, including cryo-fractionation during freezing of bottom sediments in winter, input of summer and winter precipitation, and permafrost thawing processes.