Many studies showed the impact of pesticides on diatom's metabolism. Diuron decreases the photosynthetic activity and synthesis of polyunsaturated fatty acid whereas s-metolachlor decreases the synthesis of long chain fatty acids. Fatty acids (FA) are essential elements for the structure of biological membranes and for the storage of metabolic energy. For example polyunsaturated fatty acids (PUFA) play a key role in membrane fluidity. Various studies demonstrated impacts of chemical contamination on fatty acid profiles. Furthermore, variability of fatty acid profiles of marine microalga in response to herbicides has been proved. However, to our knowledge scarce studies showed the interspecific variability of fatty acid profiles of freshwater diatoms especially in response to a pesticide contamination. The aims of this study were to investigate the interspecific variability of two freshwater diatoms on photosynthesis, growth rate, concentration in chlorophyll a and carotene and induction or repression of interest genes; Study this variability on fatty acid profiles; with and without pesticides. To address this issue, two model freshwater diatoms, Gomphonema gracile (GGRA) and Nitzschia palea (NPAL), were exposed separately during seven days to diuron and s-metolachlor at high concentration (C=10 µg/L) and at 20.5°C. For control condition, NPAL had a higher chlorophyll a and carotenoid concentration than GGRA. An interspecific variability of fatty acid profiles was observed with a lower percentage of saturated fatty acid (SFA) and highly unsaturated fatty acid (HUFA) for NPAL than GGRA. However, NPAL had a higher percentage of 22:6n3 than GGRA. Diuron decreased the percentage of chlorophyll a and carotenoid concentration of NPAL while no significant effect was observed for GGRA. Diuron increased percentage of SFA for NPAL whereas a decrease was noted for GGRA; the percentage of 14:0 and 16:0 increased for NPAL while it decreased for GGRA. NPAL had a different fatty acid profile compared to GGRA and seemed to have diverging mechanisms of protection in response to a contamination. This study proved the importance to taking into account interspecific variability of fatty acid profiles of diatoms for further use them as biomarkers of toxic contamination in fresh waters.