Increased salinity caused by saltwater intrusion or runoff from de-icing salts can severely affect freshwater vegetation and deteriorate aquatic ecosystems. These habitats can be restored with freshwater ecotypes (locally adapted populations) that tolerate above-normal salinity. Vallisneria americana is a prominent species in many freshwater ecosystems that responds differently to abiotic conditions such as substrate composition and fertility, so, in this study, we evaluated the effects of salt stress on 24 ecotypes of V. americana. Instant Ocean aquarium salt was used to create saline solutions (0.2 to 20.0 parts per thousand (ppt)), then plants were abruptly exposed to these solutions and maintained in these concentrations for five weeks before being visually assessed for quality and destructively harvested. Analysis of variance and nonlinear regression were used to calculate LC50 values—the lethal concentration of salt that reduced plant biomass and quality by 50% compared to control treatment. Growth rate and visual quality varied significantly among ecotypes, and ecotypes that were most and least sensitive to salt had 50% biomass reductions at 0.47 and 9.10 ppt, respectively. All ecotypes survived 10.0 ppt salinity concentration but none survived at 20.0 ppt, which suggests that the maximum salinity concentration tolerated by these ecotypes is between 15.0 and 20.0 ppt.