Unionid mussels are ecologically important and are globally imperiled. Toxicants contribute to mussel declines, and toxicity tests using juvenile mussels—a sensitive life stage—are valuable in determining thresholds used to set water quality criteria. In vitro culture methods provide an efficient way to propagate juveniles for toxicity testing, but their relative chemical sensitivity compared with in vivo propagated juveniles is unknown. Current testing guidelines caution against using in vitro cultured juveniles until this sensitivity is described. Our objective was to evaluate the relative sensitivity of juvenile mussels produced from both in vitro and in vivo propagation methods to selected chemicals. We conducted 96-h acute toxicity tests according to ASTM International guidelines with 3 mussel species and 6 toxicants: chloride, nickel, ammonia, and 3 copper-based compounds. Statistically significant differences between in vitro and in vivo juvenile 96-h median effect concentrations were observed in 8 of 17 tests, and in vitro juveniles were more sensitive in 6 of the 8 significant differences. At 96 h, 4 of the 8 statistically different tests for a given chemical were within a factor of 2, which is the intralaboratory variation demonstrated in a recent evaluation of mussel toxicity tests. We found that although differences in chemical sensitivity exist between in vitro and in vivo propagated juvenile mussels, they are within normal toxicity test variation. Therefore, in vitro propagated juvenile mussels may be appropriate for use in ASTM International-based toxicity testing.