In the Amazon Basin, naturally occurring methylmercury bioaccumulates in fish, which is a key source of protein consumed by riverine populations. The hydroelectric power-plant project at Santo Antônio Falls allows us to compare the Hg exposure of riverine populations sparsely distributed on both sides of the Madeira river before the area is to be flooded. From 2009 to 2011, we concluded a population survey of the area (N = 2,008; representing circa 80% of community residents) that estimated fish consumption and mercury exposure of riverine populations with different degrees of lifestyle related to fish consumption. Fish samples from the Madeira river (N = 1,615) and 110 species were analyzed for Hg. Hair-Hg was significantly lower (p < 0.001) in less isolated communities near to the capital of Porto Velho (median 2.32 ppm) than in subsistence communities in the Cuniã Lake, 180 km from Porto Velho city (median 6.3 ppm). Fish Hg concentrations ranged from 0.01 to 6.06 µg/g, depending on fish size and feeding behavior. Currently available fish in the Madeira river show a wide variability in Hg concentrations. Despite cultural similarities, riparians showed hair-Hg distribution patterns that reflect changes in fish-eating habits driven by subsistence characteristics.