Lakes, representing major freshwater resources, play a crucial role for both humans and ecosystems. Based on the increasing international interest in the contamination of water resources by genotoxic compounds, the present study aimed to evaluate the genotoxic potential of surface water samples collected from the five (5) lakes (Amvrakia, Lysimachia, Ozeros, Trichonida, Kastraki) located in Aitoloakarnania regional unit (Western Greece). The genotoxic potential of surface water samples was evaluated by employing the Cytokinesis Block MicroNucleus (CBMN) assay in cultured human lymphocytes. In the former assay, lymphocytes were treated with 1, 2 and 5% (v/v) of surface water from each lake. Statistically significant differences (1.7 to 3.3 fold increase in MN frequencies vs. the control) were seen at the dose of 5% (v/v) in all studied lakes. At the dose of 2% (v/v) statistically significant differences (1.7 to 2.6 fold increase in MN frequencies vs. the control) were observed in all studied lakes except Trichonida lake. Finally, at the dose of 1% (v/v) statistically significant differences (2.3 and 2.5 fold increase in MN frequencies vs. the control) were observed in the Ozeros and Lysimachia lakes. The evaluation of the potential genotoxic effects and the analysis of the physicochemical parameters of lakes\' surface water samples is a first step in our effort to evaluate the water quality, in terms of the presence and environmental/human risk of genotoxicants in the studied lake ecosystems. The present study showed for the first time the presence of genotoxic substances in surface waters of the studied lakes.