Freshwater mussels, one of the most imperiled groups of animals in the world, are generally under-represented in toxicity databases used for the development of ambient water quality criteria (WQC) and other environmental guidance values. Acute 96-h toxicity tests were conducted to evaluate the sensitivity of 5 species of juvenile mussels from 2 families and 4 tribes to 10 chemicals (ammonia, metals, major ions, and organic compounds), and to "screen" 10 additional chemicals (mainly organic compounds) with a commonly tested mussel species, fatmucket (Lampsilis siliquoidea). Median effect concentrations (EC50s) among the 5 species differed by: a factor of ≤ 2 for chloride, potassium, sulfate, and zinc; a factor of ≤ 5 for ammonia, chromium, copper, and nickel; and a factor of 6 and 12 for metolachlor and alachlor, indicating that mussels representing different families or tribes had similar sensitivity to most of tested chemicals regardless of modes of action. There was a strong linear relationship between EC50s for fatmucket and the other 4 mussel species across the 10 chemicals (r(2) = 0.97, slope close to 1.0), indicating that fatmucket was similar to other mussel species, and thus, this commonly tested species can be a good surrogate for protecting other mussels in acute exposures. The sensitivity of juvenile fatmucket among different populations or cultured from larvae of wild adults and captive-cultured adults was also similar in acute exposures to copper or chloride, indicating captive-cultured adult mussels can reliably be used to reproduce juveniles for toxicity testing. In compiled databases for all freshwater species, one or more mussel species were among the 4 most sensitive species for alachlor, ammonia, chloride, potassium, sulfate, copper, nickel, and zinc, and therefore, the development of WQC and other environmental guidance values for these chemicals should reflect the sensitivity of mussels. In contrast, the EC50s of fatmucket tested in the single-species study were in the high percentiles (>75(th) ) of species sensitivity distributions for 6 of 7 organic chemicals, indicating mussels might be relatively insensitive to organic chemicals in acute exposures. This article is protected by copyright. All rights reserved.