The four largest freshwater lakes in southwestern France are of both ecological and economic importance. However, some of them are subjected to mercury (Hg) contamination, resulting in the ban of human consumption of piscivorous fish. Moreover, beyond predatory fish, little information exist regarding Hg levels in other species of these ecosystems. In this context, we used a food web analytical approach to investigate Hg bioaccumulation and biomagnification in relation to the trophic structure of these four lakes. More specifically, various organisms (macrophytes, epiphyton, invertebrates and fish) were collected at the four lakes and analysed for carbon and nitrogen stable isotopes as well as for total Hg (THg) and methylmercury (MeHg). A spatial variability of bioaccumulation in organisms was observed, particularly in carnivorous fish, with higher Hg levels being found in the two more northern lakes (median±SE: 3491 ± 474 and 1113 ± 209 ng THg.g−1 dw in lakes HC and L, respectively) than in the southern pair (600 ± 117 and 911 ± 117 ng THg.g−1 dw in lakes CS and PB, respectively). Methylmercury biomagnification was observed through the food webs of all four lakes, with different trophic magnification slopes (HC = 0.16; L = 0.33; CS = 0.27; PB = 0.27), even though the length of the food chains was similar between the lakes. Our results suggest that rather than the food web structure, anthropogenic inputs (sulfate in northern lakes and phosphorus inputs in southern ones) may have a strong impact, more or less directly, on Hg methylation in freshwater environments, and lead to concentrations exceeding environmental recommendations despite low Hg backgrounds in sediment and water.Read less <