Water isotopes (δ2H and δ18O) were analyzed in samples collected in lakes associated to major riverine systems in northeastern Germany throughout 2020. This sub-dataset is derived from water samples taken at buoys temporarily installed in deep parts of the lake. Samples were taken monthly to bimonthly from March to October 2020. A Limnos water sampler was used to obtain samples from 1 m below water surface. Isotope analysis was conducted at IGB Berlin, using a Picarro L2130-i cavity ring-down spectrometer. Water temperatures were measured in similar depths with an Aqua TROLL 600 multiparameter sonde (In-Situ, Fort Collins, CO, USA). The data give information about the seasonal isotope amplitude in the sampled lakes and about spatial isotope variability in different branches of the associated riverine systems.