Shell shape variation of the freshwater mussel Castalia ambigua in relation to environmental and genetic drivers remains unresolved in South America. Along an increasing upstream to downstream hydrological gradient in the Caeté River, eastern Amazon, Brazil, we evaluated two co-occurring morphotypes of C. ambigua: the first is elongated and compressed; the second is rounded and inflated. We performed multivariate shape analysis on the lateral view of the right valve and the umbonal view, to identify potential ecophenotypic trends. We also investigated whether the degree of shell inflation was associated with sexual dimorphism. Castalia ambigua shell shape differed along the hydrological gradient and between sexes. However, in high hydraulic energy habitats the two morphotypes tend to a similar shape. Inflated shells of C. ambigua corresponded to female specimens, implying that shell shape variation in low hydraulic energy habitats was associated with sexual dimorphism. We found strong evidence of shell variation in the freshwater mussel C. ambigua associated with the environment and sexual dimorphism. Hydrological conditions appears to override sex-associated shell shape determination in high-energy habitats. Differences in shell shape and the identification of ecomorphotypes are important for management efforts, such as protecting the specific habitats associated with both morphotypes.