Water isotopes (δ2H and δ18O) were analyzed in samples collected in lakes associated to major riverine systems in northeastern Germany throughout 2020. The dataset is derived from water samples taken at a) lake shores (sampled in March and July 2020); b) buoys temporarily installed in deep parts of the lake (sampled monthly from March to October 2020); c) multiple spatially distributed spots in four selected lakes (in September 2020); d) the outflow of Müggelsee (sampled biweekly from March 2020 to January 2021). At shores, water was sampled with a pipette from 40-60 cm below water surface and directly transferred into a measurement vial, while at buoys a Limnos water sampler was used to obtain samples from 1 m below surface. Isotope analysis was conducted at IGB Berlin, using a Picarro L2130-i cavity ring-down spectrometer. 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.