An important physiological constraint influencing distributions of coastal freshwater organisms is their tolerance for saline conditions. We experimentally evaluated salinity tolerance for three freshwater mussel species (Utterbackia imbecillis, Elliptio jayensis, and Glebula rotundata). Mussels were transferred abruptly from well water to one of five treatments (0 [control], 6, 12, 18 or 24 parts per thousand [ppt]). Utterbackia imbecillis survived on average about 2 days at treatments 6 ppt, while Elliptio jayensis survived slightly longer (about 4 days). Glebula rotundata was most tolerant to salinity, surviving as well at 6 and 12 ppt as it did in the control. Additionally, G. rotundata survived at higher salinities (18 and 24 ppt) for an average of 7–8 days. To our knowledge, this is the highest salinity tolerance ever reported for a unionid. The salinity tolerance of U. imbecillis may be influenced by its inability to completely seal its valves. The variation we found in salinity tolerance of these species corresponds with their distributions in the Gulf Coastal Plain drainages: U. imbecillis and E. jayensis are primarily found in strictly freshwater habitats whereas G. rotundata inhabits lower reaches of rivers closer to the coast. Stressors such as increased frequency and intensity of storms, sea level rise, drought, low flows, fossil fuel extraction, and municipal water withdrawals, among others, may increase salinities in freshwater ecosystems, potentially stressing mussels such as U. imbecillis and E. jayensis with low salinity tolerance.