Isotope mass balance in lake systems is strongly correlated with several climatic factors such as temperature, evaporation, precipitation and air moisture. On the other hand, the sedimentary budget of lake basins driven by climate, tectonic and/or human impact is an essential pool of environmental records. Precipitation, springs and lake water δD and δ18O isotope data were used in order to understand the key factors for the hydrological balance of Balkan lakes in West Macedonia. In general, it is concluded that the open lake Ohrida and the semi-closed lake Kastoria are more buffered hydrological as karst systems and less sensitive to evaporation effect, in contrast to the closed lake system of Prespes that present a strong dependence on climate seasonality. Based on oxygen isotopes in bulk sediments, it is concluded that in Kastoria lake basin an increased run-off on the land surface, probably under a more humid period, in 2.4 kyr BP disturbed the transition to a drier regime from mid/late Holocene to present.